Anti-Viral Azide Containing Compounds

ABSTRACT

Methods of using azide-modified biomolecules, such as fatty acids, carbohydrates and lipids, to treat a plant, an insect or an animal infected with a virus or to inhibit infectivity of a virus, such as the human immunodeficiency virus, are provided. Also provided are methods of labeling a virus, such as human immunodeficiency virus, with an azide-modified biomolecule, such as a fatty acid, a carbohydrate, or an isoprenoid lipid. Also, provided are methods of tracking a virus in vivo, with an azide-modified biomolecule, such as a fatty acid, a carbohydrate, or an isoprenoid lipid. The azide-modified biomolecules may be combined with a pharmaceutically acceptable excipient to produce a pharmaceutical composition, optionally containing another anti-viral agent and/or a delivery agent, such as a liposome.

BACKGROUND

Viral infections account for significant morbidity and mortality inhumans and animals. In addition, viral infections also result insignificant agricultural losses, with plant viruses causing an estimated$60 billion in lost crop yields throughout the world each year. Althoughsignificant resources have been dedicated to identifying compoundshaving anti-viral properties, viral infections continue to present asignificant risk to human health and agriculture.

In addition, the usefulness of most existing anti-viral treatments islimited by the development of multidrug resistance, poor efficacy,and/or toxicity. In fact, many anti-viral treatments are highly toxicand can cause serious side effects, including heart damage, kidneyfailure and osteoporosis. Other challenges include creating a drug thatis broadly applicable in combating many different types of viralinfections, which can be particularly important in the treatment ofimmunocompromised individuals.

One virus in particular, the human immunodeficiency virus (HIV), remainsa global pandemic despite the development of antiretroviral drugstargeting HIV. As of 2007, it was estimated that more than 33 millionpeople were infected with HIV, and HIV associated diseases represent amajor world health problem. HIV is a retrovirus that infects CD4⁺ cellsof the immune system, destroying or impairing their function. As theinfection progresses, the immune system becomes weaker, leaving theinfected person more susceptible to opportunistic infections and tumors,such as Kaposi's sarcoma, cervical cancer, lymphoma, and neurologicaldisorders. The most advanced stage of HIV infection is acquiredimmunodeficiency syndrome (AIDS). It can take 10-15 years for anHIV-infected person to develop AIDS. Certain antiretroviral drugs candelay the process even further.

Although much effort has been put forth into designing effectivetherapeutics against HIV, currently no curative anti-retroviral drugsagainst HIV exist. Several stages of the HIV life cycle have beenevaluated as targets for the development of therapeutic agents (Mitsuya,H. et al., 1991, FASEB J 5:2369-2381). One area of focus has been theHIV reverse transcriptase enzyme. Reverse transcriptase copies the HIV,single stranded RNA genome into double-stranded viral DNA. The viral DNAis then integrated into the host's chromosomal DNA where the host'scellular processes, like transcription and translation, are used toproduce viral proteins and ultimately new virus particles. Therefore,interfering with reverse transcriptase inhibits HIV's ability toreplicate. One class of reverse transcriptase inhibitors is nucleosideanalogs, such as Zidovudine (AZT), Didanosine (ddI), Zalcitabine (ddC),and Stavudine (d4T), Lamivudine (3TC), Abacavir (ABC), Emtricitabine(FTC), Entecavir (INN), and Apricitabine (ATC) (Mitsuya, H. et al.,1991, Science 249:1533-1544; El Kouni, Curr Pharm Des, 2002, 8:581-93;Sharma et al., Cur Top Med Chem, 2004, 4:895-919). Another class ofreverse transcriptase inhibitors is nucleotide analogs, such asTenofovir (tenofovir disoproxil fumarate) and Adefovir (bis-POM PMPA)(Palmer et al., AIDS Res Hum Retroviruses, 2001, 17:1167-73). Thesenucleoside and nucleotide compounds are analogs of the naturallyoccurring deoxyribose nucleotides, however, the analogs lack the3′-hydroxyl group on the deoxyribose sugar. As a result, when theanalogs are incorporated into a growing viral DNA chain, the incomingdeoxynucleotide cannot form a phosphodiester bond with the analog thatis needed to extend the DNA chain. Thus, the analogs terminate viral DNAreplication. Another class of reverse transcriptase inhibitors is thenon-nucleoside reverse transcriptase inhibitors, such as Efavirenz,Nevirapine, Delavirdine, and Etravirine (El Safadi et al., ApplMicrobiol Biotechnol, 2007, 75:723-37). They have a different mode ofaction than the nucleoside and nucleotide inhibitors, binding to thereverse transcriptase and interfering with its function.

The late stages of HIV replication involve processing of certain viralproteins prior to the final assembly of new virions. This late-stageprocessing is dependent, in part, on the activity of a viral protease.Thus, another area of focus in the development of antiretroviral drugsis protease inhibitors, such as saquinavir, ritonavir, indinavir,nelfinavir, amprenavir, lopinavir, and atazanavir (Erickson, J., 1990,Science 249:527-533; Klei et al., J Virol, 81:9525-35).

Other antiretroviral drugs target viral entry into the cell, theearliest stage of HIV infection. For HIV to enter a cell, its surfacegp120 protein binds to CD4, exposing a conserved region of gp120 thatbinds to a CCR5 or a CXCR4 co-receptor. After gp120 binds to theco-receptor, a hydrophobic fusion peptide at the N-terminus of the gp41envelope protein is exposed and inserted into the membrane of the cell.Entry inhibitors work by interfering with any stage of the viral entryprocess. For example, recombinant soluble CD4, for example, has beenshown to inhibit infection of CD-4⁺ T-cells by some HIV-1 strains(Smith, D. H. et al., 1987, Science 238:1704-1707). Similarly, TNX-355is a monoclonal antibody that binds CD4 and inhibits binding to gp120(Kuritzkes et al., J Infect Dis, 2004, 189:286-91). BMS-806 binds to theviral envelope protein and inhibits binding to CD4 (Veazy et al., Nature2003, 438:99-102). Co-receptor binding can be inhibited by several CCR5inhibitors, including SCH-C and SCH-D, UK-427,857, maraviroc,vicriviroc, and an anti-CCR5 antibody (PRO-140) (Emmelkamp et al., Eur JMed Res, 2007, 12:409-17). Co-receptor binding can also be inhibited bythe CXCR4 inhibitors AMD3100 and AMD070 (De Clerq, Nature Reviews DrugDiscovery 2003, 2:581-87). Other compounds, such as enfuvirtide, bind togp41 and interfere with its ability to mediate membrane fusion and entry(La Bonte et al., Nature Reviews Drug Discovery 2003, 2:345-36).

While beneficial, these antiretroviral drugs often exhibit toxic sideeffects such as bone marrow suppression, vomiting, and liver functionabnormalities. In addition, they are not curative, probably due to therapid appearance of drug resistant HIV mutants (Lander, B. et al., 1989,Science 243:1731-1734). Drug-resistant HIV strains develop due to thevery high genetic variability of HIV. This genetic variability resultsfrom several factors, including HIV's fast replication cycle, with thegeneration of 10⁹ to 10¹⁰ virions per day, a high mutation rate ofapproximately 3×10⁻⁵ per nucleotide base per cycle of replication, andrecombinogenic properties of reverse transcriptase.

To combat the development of drug resistant HIV strains, multiple drugshave been combined as part of highly active antiretroviral therapy(HAART) (El Safadi et al., Appl Microbiol Biotechnol, 2007, 75:723-37;Sharma et al., Cur Top Med Chem, 2004, 4:895-919). Currently HAARTtypically involves combining at least three drugs belonging to at leasttwo classes of antiretroviral agents. As discussed above, these classesinclude nucleoside or nucleotide analog reverse transcriptaseinhibitors, non-nucleoside reverse transcriptase inhibitors, proteaseinhibitors, and entry inhibitors.

Thus, although a great deal of effort is being directed to the designand testing of anti-viral drugs, the search for new and improved methodsof treating viral infections, such as HIV, continues.

SUMMARY

The present disclosure provides methods of using azide-modifiedbiomolecules, such as azide-modified fatty acids, azide-modifiedcarbohydrates, azide-modified isoprenoid lipids, or pharmaceuticallyacceptable salt thereof for treating viral infections, such as HIVinfections, or for labeling a protein of a virus, such as HIV, as wellas pharmaceutical compositions containing an azide-modified biomoleculeor pharmaceutically acceptable salt thereof.

One aspect of the disclosure is directed to a method of treating asubject infected with a plant, an insect, or an animal virus and in needof treatment for the infection, the method comprising administering tothe subject a therapeutically effective amount of an azide-modifiedfatty acid, an azide-modified carbohydrate, an azide-modified isoprenoidlipid or pharmaceutically acceptable salt thereof.

In some embodiments, the azide-modified fatty acid or pharmaceuticallyacceptable salt thereof has the formula:

Y—CH₂—X—CO₂H  [I]

wherein,

Y is H or an azido group; and

when Y is an azido group, X is a linear or branched carbon chaincomprising 6 to 28 carbons, wherein one or more of said carbons may beindependently replaced by an oxygen, selenium, silicon, sulfur, SO, SO₂or NR₁, or wherein one or more pairs of said carbons adjacent to oneanother may be attached to one another by a double or triple bond; or

when Y is H, X is a linear or branched carbon chain comprising 6 to 28carbons, wherein one hydrogen on one of said carbons is replaced with anazido group and wherein one or more of said carbons not having an theazido group attached thereto may be independently replaced by an oxygen,selenium, silicon, sulfur, SO, SO₂ or NR₁, or wherein one or more pairsof said carbons adjacent to one another and not having an azido groupmay be attached to one another by a double or triple bond;

wherein, R₁ is H or an alkyl comprising 1 to 6 carbons.

In some of these embodiments, Y is an azido group. In some of these, Xis a linear carbon chain. In some of these, the linear carbon chaincomprises 8 to 15 carbons. In some of these, the linear carbon chaindoes not contain an oxygen, selenium, silicon, sulfur, SO, SO₂ or NR₁.In some of these, the carbon chain does not contain a double or triplebond. In some of these, the azide modified fatty acid is15-azidopentadecanoic acid, 12-azidododecanoic acid, or pharmaceuticallyacceptable salt thereof.

In some embodiments, the azide-modified carbohydrate is an N-linkedcarbohydrate or an O-linked carbohydrate. In some embodiments, theazide-modified carbohydrate is tetraacetylatedN-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine,or tetraacetylated N-azidoacetylglucosamine.

In some embodiments, the azide-modified isoprenoid lipid comprises afarnesyl group or a geranylgeranyl group. In some of these, theazide-modified isoprenoid lipid is an azido farnesyl diphosphate, anazido farnesyl alcohol, an azido geranylgeranyl diphosphate, or an azidogeranylgeranyl alcohol.

In some embodiments, the virus is an non-human animal virus or a humananimal virus.

In some embodiments, the non-human animal virus is a picornavirus, apestivirus, an arterivirus, a coronavirus, a paramyxovirus, anorthomyxovirus, a reovirus, a porcine, a circovirus, a herpesvirus, anasfarvirus, a retrovirus, a flavivirus, or a rhabdovirus.

In some of embodiments, the human animal virus is an adenovirus, anastrovirus, a hepadnavirus, a herpesvirus, a papovavirus, a poxvirus, anarenavirus, a bunyavirus, a calcivirus, a coronavirus, a filovirus, aflavivirus, an orthomyxovirus, a paramyxovirus, a picornavirus, areovirus, a retrovirus, a rhabdovirus, or a togavirus. In some of these,the retrovirus is a human immunodeficiency virus or a humanT-lymphotrophic virus. In some of these, the retrovirus is the humanimmunodeficiency virus, HIV-1.

In some embodiments, the virus is a plant virus. In some of these, theplant virus is an alfamovirus, an allexivirus, an alphacryptovirus, ananulavirus, an apscaviroid, an aureusvirus, an avenavirus, anaysunviroid, a badnavirus, a begomovirus, a benyvirus, abetacryptovirus, a betaflexiviridae, a bromovirus, a bymovirus, acapillovirus, a carlavirus, a carmovirus, a caulimovirus, a cavemovirus,a cheravirus, a closterovirus, a cocadviroid, a coleviroid, a comovirus,a crinivirus, a cucumovirus, a curtovirus, a cytorhabdovirus, adianthovirus, an enamovirus, an umbravirus & B-type satellite virus, afabavirus, a fijivirus, a furovirus, a hordeivirus, a hostuviroid, anidaeovirus, an ilarvirus, an ipomovirus, a luteovirus, a machlomovirus,a macluravirus, a marafivirus, a mastrevirus, a nanovirus, a necrovirus,a nepovirus, a nucleorhabdovirus, an oleavirus, an ophiovirus, anoryzavirus, a panicovirus, a pecluvirus, a petuvirus, a phytoreovirus, apolerovirus, a pomovirus, a pospiviroid, a potexvirus, a potyvirus, areovirus, a rhabdovirus, a rymovirus, a sadwavirus, a SbCMV-like virus,a sequivirus, a sobemovirus, a tenuivirus, a TNsatV-like satellitevirus, a tobamovirus, a topocuvirus, a tospovirus, a trichovirus, atritimovirus, a tungrovirus, a tymovirus, an umbravirus, avaricosavirus, a vitivirus, or a waikavirus.

In some embodiments, the virus is an insect virus. In some of these, theinsect virus is a densovirus, an iridovirus, a chloriridovirus, abaculovirus, a polydnavirus, an entomopox virus, an ascovirus, an insectpicornavirus, a calicivirus, or a nodavirus.

In some embodiments, the subject is a human animal.

Another aspect of the disclosure is directed a method of inhibiting theinfectivity of a virus, the method comprising contacting a cell infectedwith the virus with an azide-modified fatty acid, an azide-modifiedcarbohydrate, an azide-modified isoprenoid lipid, or pharmaceuticallyacceptable salt thereof in an amount effective to inhibit theinfectivity of the virus.

In some embodiments, the azide-modified fatty acid or pharmaceuticallyacceptable salt thereof has the Formula [I], as described above. In someof these, Y is an azido group. In some of these, X is a linear carbonchain. In some of these, the linear carbon chain comprises 8 to 15carbons. In some of these, the linear carbon chain does not contain anoxygen, selenium, silicon, sulfur, SO, SO₂ or NR₁. In some of these, thecarbon chain does not contain a double or triple bond. In some of these,the azide modified fatty acid is 15-azidopentadecanoic acid,12-azidododecanoic acid, or pharmaceutically acceptable salt thereof.

In some embodiments, the azide-modified carbohydrate is an N-linkedcarbohydrate or an O-linked carbohydrate. In some embodiments, theazide-modified carbohydrate is tetraacetylatedN-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine,or tetraacetylated N-azidoacetylglucosamine.

In some embodiments, the azide-modified isoprenoid lipid comprises afarnesyl group or a geranylgeranyl group. In some of these, theazide-modified isoprenoid lipid is an azido farnesyl diphosphate, anazido farnesyl alcohol, an azido geranylgeranyl diphosphate, or an azidogeranylgeranyl alcohol.

In some embodiments, the virus is a non-human animal virus or humananimal virus.

In some embodiments, the virus is a non-human animal virus. In someembodiments, the non-human animal virus is a picornavirus, a pestivirus,an arterivirus, a coronavirus, a paramyxovirus, an orthomyxovirus, areovirus, a porcine, a circovirus, a herpesvirus, an asfarvirus, aretrovirus, a flavivirus, or a rhabdovirus.

In some of embodiments, the virus is a human animal virus. In some ofthese, the human animal virus is an adenovirus, an astrovirus, ahepadnavirus, a herpesvirus, a papovavirus, a poxvirus, an arenavirus, abunyavirus, a calcivirus, a coronavirus, a filovirus, a flavivirus, anorthomyxovirus, a paramyxovirus, a picornavirus, a reovirus, aretrovirus, a rhabdovirus, or a togavirus. In some of these, theretrovirus is a human immunodeficiency virus or a human T-lymphotrophicvirus. In some of these, the retrovirus is the human immunodeficiencyvirus, HIV-1.

In some embodiments, the virus is a plant virus. In some of these, theplant virus is an alfamovirus, an allexivirus, an alphacryptovirus, ananulavirus, an apscaviroid, an aureusvirus, an avenavirus, anaysunviroid, a badnavirus, a begomovirus, a benyvirus, abetacryptovirus, a betaflexiviridae, a bromovirus, a bymovirus, acapillovirus, a carlavirus, a carmovirus, a caulimovirus, a cavemovirus,a cheravirus, a closterovirus, a cocadviroid, a coleviroid, a comovirus,a crinivirus, a cucumovirus, a curtovirus, a cytorhabdovirus, adianthovirus, an enamovirus, an umbravirus & B-type satellite virus, afabavirus, a fijivirus, a furovirus, a hordeivirus, a hostuviroid, anidaeovirus, an ilarvirus, an ipomovirus, a luteovirus, a machlomovirus,a macluravirus, a marafivirus, a mastrevirus, a nanovirus, a necrovirus,a nepovirus, a nucleorhabdovirus, an oleavirus, an ophiovirus, anoryzavirus, a panicovirus, a pecluvirus, a petuvirus, a phytoreovirus, apolerovirus, a pomovirus, a pospiviroid, a potexvirus, a potyvirus, areovirus, a rhabdovirus, a rymovirus, a sadwavirus, a SbCMV-like virus,a sequivirus, a sobemovirus, a tenuivirus, a TNsatV-like satellitevirus, a tobamovirus, a topocuvirus, a tospovirus, a trichovirus, atritimovirus, a tungrovirus, a tymovirus, an umbravirus, avaricosavirus, a vitivirus, or a waikavirus.

In some embodiments, the virus is an insect virus. In some of these, theinsect virus is a densovirus, an iridovirus, a chloriridovirus, abaculovirus, a polydnavirus, an entomopox virus, an ascovirus, an insectpicornavirus, a calicivirus, or a nodavirus.

In some embodiments, the cell is a human cell.

A third aspect of the disclosure is directed to a method of producing avirus labeled with an azide-modified fatty acid, an azide-modifiedcarbohydrate, an azide-modified isoprenoid lipid, or a pharmaceuticallyacceptable salt thereof, the method comprising contacting a cellinfected with the virus with the azide-modified fatty acid, theazide-modified carbohydrate, the azide-modified isoprenoid lipid, orpharmaceutically acceptable salt thereof so that the azide-modifiedfatty acid, the azide-modified carbohydrate, the azide-modifiedisoprenoid lipid, or pharmaceutically acceptable salt thereof enters thecell and is incorporated into a protein of the virus, thereby producingthe labeled virus.

In some embodiments, the method is a method of producing a humanimmunodeficiency virus labeled with an azide-modified fatty acid, anazide-modified carbohydrate, an azide-modified isoprenoid lipid, orpharmaceutically acceptable salt thereof, the method comprisingcontacting a cell infected with the human immunodeficiency virus withthe azide-modified fatty acid, the azide-modified carbohydrate, theazide-modified isoprenoid lipid, or pharmaceutically acceptable saltthereof so that the azide-modified fatty acid, the azide-modifiedcarbohydrate, the azide-modified isoprenoid lipid, or pharmaceuticallyacceptable salt thereof enters the cell and is incorporated into aprotein of the virus, thereby producing the labeled virus.

In some embodiments, the azide-modified fatty acid or pharmaceuticallyacceptable salt thereof has the Formula [I], as described above. In someof these, Y is an azido group. In some of these, X is a linear carbonchain. In some of these, the linear carbon chain comprises 8 to 15carbons. In some of these, the linear carbon chain does not contain anoxygen, selenium, silicon, sulfur, SO, SO₂ or NR₁. In some of these, thecarbon chain does not contain a double or triple bond. In some of these,the azide modified fatty acid is 15-azidopentadecanoic acid,12-azidododecanoic acid, or pharmaceutically acceptable salt thereof.

In some embodiments, the azide-modified carbohydrate is an N-linkedcarbohydrate or an O-linked carbohydrate. In some embodiments, theazide-modified carbohydrate is tetraacetylatedN-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine,or tetraacetylated N-azidoacetylglucosamine.

In some embodiments, the azide-modified isoprenoid lipid comprises afarnesyl group or a geranylgeranyl group. In some of these, theazide-modified isoprenoid lipid is an azido farnesyl diphosphate, anazido farnesyl alcohol, an azido geranylgeranyl diphosphate, or an azidogeranylgeranyl alcohol.

In some embodiments, the cell is a human cell.

In some embodiments, the virus is a human immunodeficiency virus, whilein others, the virus is a baculovirus.

In some embodiments, the azide-modified carbohydrate, azide-modifiedfatty acid, azide-modified isoprenoid lipid, or pharmaceuticallyacceptable salt thereof is formulated with a pharmaceutically acceptableexcipient.

In some embodiments, the method further comprises the step ofadministering to the cell the azide-modified carbohydrate,azide-modified fatty acid, azide-modified isoprenoid lipid, orpharmaceutically acceptable salt thereof which is formulated with apharmaceutically acceptable excipient.

A fourth aspect of the disclosure is directed to a method of tracking avirus in vivo comprising the steps of contacting cultured cells or asubject with an azide-modified carbohydrate, azide-modified fatty acid,azide-modified isoprenoid lipid, or a pharmaceutically acceptable saltthereof; contacting the cultured cells or the subject with an alkynelabeled reporter molecule; and tracking the reporter-labeled virus inthe cultured cells or the subject.

In some embodiments, the cultured cells or the subject is contacted withan azide-modified fatty acid or pharmaceutically acceptable saltthereof.

In some embodiments, the azide-modified fatty acid or pharmaceuticallyacceptable salt thereof has the Formula [I], as described above. In someembodiments, Y is an azido group. In some of these, X is a linear carbonchain. In some of these, the linear carbon chain comprises 8 to 15carbons. In some of these, the linear carbon chain does not contain anoxygen, selenium, silicon, sulfur, SO, SO₂ or NR₁. In some of these, thecarbon chain does not contain a double or triple bond. In some of these,the azide modified fatty acid is 15-azidopentadecanoic acid,12-azidododecanoic acid, or pharmaceutically acceptable salt thereof.

Yet another aspect of the disclosure is directed to a pharmaceuticalcomposition comprising an azide-modified fatty acid, an azide-modifiedcarbohydrate, an azide-modified isoprenoid lipid, or pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable excipient.

In some embodiments, the azide-modified fatty acid or pharmaceuticallyacceptable salt thereof has the Formula [I], as described above. In someof these, Y is an azido group. In some of these, X is a linear carbonchain. In some of these, the linear carbon chain comprises 8 to 15carbons. In some of these, the linear carbon chain does not contain anoxygen, selenium, silicon, sulfur, SO, SO₂ or NR₁. In some of these, thecarbon chain does not contain a double or triple bond. In some of these,the azide modified fatty acid is 15-azidopentadecanoic acid,12-azidododecanoic acid, or pharmaceutically acceptable salt thereof.

In some embodiments, the azide-modified carbohydrate is an N-linkedcarbohydrate or an O-linked carbohydrate. In some embodiments, theazide-modified carbohydrate is tetraacetylatedN-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine,or tetraacetylated N-azidoacetylglucosamine.

In some embodiments, the azide-modified isoprenoid lipid comprises afarnesyl group or a geranylgeranyl group. In some of these, theazide-modified isoprenoid lipid is an azido farnesyl diphosphate, anazido farnesyl alcohol, an azido geranylgeranyl diphosphate, or an azidogeranylgeranyl alcohol.

In some embodiments, the composition further comprises at least oneanti-viral agent. In some of these, the anti-viral agent is selectedfrom a reverse transcriptase inhibitor, a viral protease inhibitor, aviral fusion inhibitor, a viral integrase inhibitor, a glycosidaseinhibitor, a viral neuraminidase inhibitor, an M2 protein inhibitor, anamphotericin B, hydroxyurea, α-interferon, β-interferon, γ-interferon,and an antisense oligonucleotide. In some of these, the reversetranscriptase inhibitor is at least one of Zidovudine (AZT), Didanosine(ddI), Zalcitabine (ddC), ddA, Stavudine (d4T), Lamivudine (3TC),Abacavir (ABC), Emtricitabine (FTC), Entecavir (INN), Apricitabine(ATC), Atevirapine, ribavirin, acyclovir, famciclovir, valacyclovir,ganciclovir, valganciclovir, Tenofovir, Adefovir, PMPA, cidofovir,Efavirenz, Nevirapine, Delavirdine, or Etravirine; wherein the virusprotease inhibitor is at least one of tipranavir, darunavir, indinavir,lopinavir, fosamprenavir, atazanavir, saquinavir, ritonavir, indinavir,nelfinavir, or amprenavir; wherein the viral fusion inhibitor is atleast one of a CD4 antagonist, a CCR5 antagonist, a CXCR4 antagonist, orenfuvirtide; wherein the viral integrase is raltegravir; wherein theglycosidase inhibitor is at least one of SC-48334 or MDL-28574; whereinthe viral neuraminidase inhibitor is at least one of oseltamivir,peramivir, zanamivir, and laninamivir; and wherein the M2 proteininhibitor is at least one of amantadine or rimantidine.

In some embodiments, the composition further comprises an agent fordelivering the azide-modified fatty acid, the azide-modifiedcarbohydrate, the azide-modified isoprenoid lipid or pharmaceuticallyacceptable salt thereof to a cell. In some of these, an agent fordelivering the azide-modified fatty acid, the azide-modifiedcarbohydrate, or the azide-modified isoprenoid lipid to a cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate certain embodiments of theinvention, and together with the written description, serve to explaincertain principles of the invention.

FIG. 1 shows a time course of azide-modified proteins in HIV-infectedCEMx174 cells. CEMx174 infected cells were labeled with either (A)15-azidopentadecanoic acid, (B) 12-azidododecanoic acid, (C)tetraacetylated N-azidoacetylgalactosamine, or (D) tetraacetylatedN-azidoacetyl-D-mannosamine and were harvested at 12, 24, 72 hours, and14 days post-infection. FIG. 1(E) shows a representative gel that waspost-stained with the total protein stain: SYPRO® Ruby protein stain(Sigma-Aldrich, St. Louis, Mo.).

FIG. 2 shows gel electrophoresis of azide-modified proteins from HIVproduced from chronically infected CEMx174 cells. FIG. 2(A) shows viralproteins tagged with tetraacetylated N-azidoacetyl-D-mannosamine (Man),tetraacetylated N-azidoacetylgalactosamine (GalNaz),15-azidopentadecanoic acid (Palmitic), 12-azidododecanoic acid(Myristic) and labeled with TAMRA. FIG. 2(B) shows a total protein stainusing SYPRO® Ruby protein stain (Sigma-Aldrich, St. Louis, Mo.).

FIG. 3 shows the results of a luciferase reporter assay (AppliedBiosytems luciferase reagent) to measure the infectivity of unlabeledHIV (CONTROL) or HIV labeled with 15-azidopentadecanoic acid (PALM),12-azidododecanoic acid (MYR), tetraacetylatedN-azidoacetyl-D-mannosamine (MAN), or tetraacetylatedN-azidoacetylgalactosamine (GAL).

FIG. 4 shows the results of a luciferase reporter assay (Promegaluciferase reagent) to measure the infectivity of unlabeled HIV(CONTROL) or HIV labeled with 15-azidopentadecanoic acid (PALM),12-azidododecanoic acid (MYR), tetraacetylatedN-azidoacetyl-D-mannosamine (MAN), or tetraacetylatedN-azidoacetylgalactosamine (GAL).

FIG. 5 shows the results of post translational modification (PTM) analogincorporation on the ability of BacMam to enter mammalian cell. Thepanels show both phase (bottom row of panels) and fluorescent GFP images(top row of panels) of U2-OS cells infected with PTM analog-labeledBacMam viruses.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. In order that the presentinvention may be more readily understood, certain terms are firstdefined. Additional definitions are set forth throughout the detaileddescription. In case of conflict, the present specification, includingdefinitions, will control.

As used herein, “azide-modified fatty acid” refers to a fatty acid thatcomprises an azido group and has the following formula, R—N₃ where Rcomprises a hydrocarbon chain with at least one carboxylic acidfunctional group, which is usually, although not necessarily, at aterminal position.

As used herein, “azide-modified carbohydrate” refers to a carbohydratethat comprises an azido group and has the following formula, R—N₃ whereR is a carbohydrate.

As used herein, “azide-modified isoprenoid lipid” refers to anisoprene-containing lipid, or derivative thereof. The azide-modifiedisoprenoid comprises an azido group and has the following formula, R—N₃where R is an isoprene-containing lipid, such as the C₁₅ farnesylisoprenoid lipid or the C₂₀ geranylgeranyl isoprenoid lipid, or aderivative thereof, including, but not limited to, an azido farnesyldiphosphate, an azido farnesyl alcohol, an azido geranylgeranyldiphosphate, or an azido geranylgeranyl alcohol.

As used herein, “animal virus” refers to a virus that infects anon-human animal or human animal cell. A non-human animal virus infectsnon-human animal cells. In certain instances, a virus that infectsnon-human animal cells is also capable of infecting human animal cells.A human animal virus infects human animal cells. In certain instances, avirus that infects human animal cells is also capable of infectingnon-human animal cells.

As used herein, “biomolecule,” refers to proteins, peptides, aminoacids, glycoproteins, nucleic acids, nucleotides, nucleosides,oligonucleotides, sugars, oligosaccharides, lipids, hormones,proteoglycans, carbohydrates, polypeptides, polynucleotides,polysaccharides, which having characteristics typical of molecules foundin living organisms and may be naturally occurring or may be artificial(not found in nature and not identical to a molecule found in nature).

As used herein, “click chemistry,” refers to the copper(I)-catalyzedvariant of the Huisgen cycloaddition or the 1,3-dipolar cycloadditionbetween an azide and a terminal alkyne to form a 1,2,4-triazole. Suchchemical reactions can use, but are not limited to, simple heteroatomicorganic reactants and are reliable, selective, stereospecific, andexothermic.

As used herein, “cycloaddition” refers to a chemical reaction in whichtwo or more π (pi)-electron systems (e.g., unsaturated molecules orunsaturated parts of the same molecule) combine to form a cyclic productin which there is a net reduction of the bond multiplicity. In acycloaddition, the π (pi) electrons are used to form new π (pi) bonds.The product of a cycloaddition is called an “adduct” or “cycloadduct”.Different types of cycloadditions are known in the art including, butnot limited to, [3+2]cycloadditions and Diels-Alder reactions.[3+2]cycloadditions, which are also called 1,3-dipolar cycloadditions,occur between a 1,3-dipole and a dipolarophile and are typically usedfor the construction of five-membered heterocyclic rings. The term“[3+2]cycloaddition” also encompasses “copperless” [3+2]cycloadditionsbetween azides and cyclooctynes and difluorocyclooctynes described byBertozzi et al. J. Am. Chem. Soc., 2004, 126:15046-15047.

As used herein, “DNA virus” refers to a virus that has deoxyribonucleicacid (DNA) as its genetic material. DNA viruses are usually doublestranded but may also be single stranded.

As used herein, “glycoprotein” refers to a protein that has beenglycosylated and those that have been enzymatically modified, in vivo orin vitro, to comprise a carbohydrate group.

As used herein, “HIV” and “human immunodeficiency virus” refer to humanimmunodeficiency virus 1 and 2 (HIV-1 and HIV-2).

As used herein, “infectivity” refers to the ability of a virus to enteror exit a cell.

As used herein, “insect virus” refers to a virus that infects insectcells. Certain insect viruses, such as, for example, unmodifiedbaculovirus or modified baculovirus (BacMam), can also infect non-humananimal and/or human animal cells.

As used herein, “plant virus” refers to a virus that infects plantcells.

As used herein, “pharmaceutically acceptable excipient” includessolvents, dispersion media, diluents, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, etc., thatare compatible with pharmaceutical administration. Use of these agentsfor pharmaceutically active substances is well known in the art.

As used herein, “protein” and “polypeptide” are used in a generic senseto include polymers of amino acid residues of any length. The term“peptide” is used herein to refer to polypeptides having less than 100amino acid residues, typically less than 10 amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues are an artificial chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers.

As used herein, “reporter molecule” refers to any moiety capable ofbeing attached to a modified post translationally modified protein ofthe present invention, and detected either directly or indirectly.Reporter molecules include, without limitation, a chromophore, afluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye,a particle, a hapten, an enzyme and a radioisotope. Preferred reportermolecules include fluorophores, fluorescent proteins, haptens, andenzymes.

As used herein, “RNA virus” refers to a virus that has ribonucleic acid(RNA) as its genetic material. RNA viruses are usually single strandedbut may also be double stranded.

As used herein, the term “subject” is intended to include human andnon-human animals, plants, and insects. Subjects may include a humanpatient having a viral infection, including, but not limited to, an HIVinfection. The term “non-human animals” of the invention includes allvertebrates, such as non-human primates, sheep, dogs, cats, cows, goats,horses, chickens, pigs, amphibians, reptiles, etc.

As used herein, “treatment” or “treating” refers to a therapeutic orpreventative measure. The treatment may be administered to a subjecthaving having a disorder which may include, but is not limited to, amedical disorder in the case where the subject is an animal, or whoultimately may acquire the disorder, in order to prevent, cure, delay,reduce the severity of, and/or ameliorate one or more symptoms of adisorder or recurring disorder, or in order to prolong the survival of asubject beyond that expected in the absence of such treatment.

As used herein, a “therapeutically effective amount” or “effectiveamount” means the amount of a compound that, when administered to anon-human animal or human animal, a plant, an insect, or other subjectfor treating a disease, is sufficient to effect such treatment for thedisease. The “effective amount” will vary depending on the compound, thedisease and its severity and the age, weight, etc., of the subject to betreated.

It must be noted that, as used in this specification and the appendedclaims, the singular form “a”, “an” and “the” include plural referentsunless the context dictates otherwise. Thus, for example, reference to“a virus” includes a plurality of viruses unless the context dictatesotherwise.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. In addition, the materials, methods,and examples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

DETAILED DESCRIPTION

The present disclosure concerns the use of azide-modified biomolecules,such as fatty acids or carbohydrates, for treating viral infections, aswell as pharmaceutical compositions containing an azide-modifiedbiomolecule. Azide-modified fatty acids, azide-modified carbohydrates,and azide-modified isoprenoid lipids have been previously described asuseful reagents for labeling and detecting proteins of interest as partof a click chemistry reaction involving a copper (I)-catalyzedcycloaddition reaction between an azide and an alkyne. See CLICK-IT®metabolic labeling reagents for proteins (Invitrogen, Carlsbad, Calif.);see also, U.S. Patent Application Publication No. 2007/0249014 and U.S.Patent Application Publication No. 20050222427, which disclosures arehereby incorporated by reference in its entirety. Applicants, however,have unexpectedly discovered that these azide-modified biomolecules haveanti-viral activity and can be used to treat viral infections. It wassurprisingly discovered that these azide-modified biomoleculesprofoundly affect viral infectivity and that labeling viruses with theseazide-modified biomolecules inhibited viral entry into host cells.Without intending to be bound by any theory, it appears thatpost-translational modification of viral proteins with an azido-modifiedbiomolecule at sites normally occupied by unmodified biomolecules, suchas saturated fatty acids (e.g., myristic acid and palmitic acid), mayresult in the inhibition of infectivity of the virus in a manner similarto the absence of these biomolecules at these sites.

Click Chemistry

Azides and terminal or internal alkynes can undergo a 1,3-dipolarcycloaddition (Huisgen cycloaddition) reaction to give a 1,2,3-triazole.However, this reaction requires long reaction times and elevatedtemperatures. Alternatively, azides and terminal alkynes can undergoCopper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) at roomtemperature. Such copper(I)-catalyzed azide-alkyne cycloadditions, alsoknown as click chemistry, is a variant of the Huisgen 1,3-dipolarcycloaddition wherein organic azides and terminal alkynes react to give1,4-regioisomers of 1,2,3-triazoles. Examples of click chemistryreactions are described by Sharpless et al. (U.S. Patent ApplicationPublication No. 20050222427, PCT/US03/17311; Lewis W G, et al.,Angewandte Chemie-Int'l Ed. 41 (6): 1053; method reviewed in Kolb, H.C., et al., Angew. Chem. Inst. Ed. 2001, 40:2004-2021), which developedreagents that react with each other in high yield and with few sidereactions in a heteroatom linkage (as opposed to carbon-carbon bonds) inorder to create libraries of chemical compounds.

Click chemistry has been used to label and detect proteins of interest.For example, the CLICK-IT® (Invitrogen, Carlsbad, Calif.) reaction is atwo-step labeling technique involving the incorporation of a modifiedmetabolic precursor, such as an azide-modified fatty acid, anazide-modified carbohydrate, or an azide-modified isoprenoid lipid, intoproteins as a chemical “handle” followed by the chemoselective ligation(or “click” reaction) between an azide and an alkyne. In the clickreaction, the modified protein is detected with a corresponding azide-or alkyne-containing dye or hapten. The CLICK-IT® metabolic labelingreagents have been used to monitor post translational modifications ofproteins, such as acylation, glycosylation, and prenylation, andinclude 1) azide-modified fatty acids, such as CLICK-IT® palmitic acidazide (i.e., 15-azidopentadecanoic acid) and CLICK-IT® myristic acidazide (i.e., 12-azidododecanoic acid), for labeling palmitoylated andmyristoylated proteins, respectively; 2) azide-modified carbohydrates,including CLICK-IT® GalNAz (tetraacetylated N-azidoacetylgalactosamine)for labeling O-linked glycoproteins, CLICK-IT® ManNAz (tetraacetylatedN-azidoacetyl-D-mannosamine) for labeling sialic acid modifiedglycoproteins, and CLICK-IT® GlcNAz (tetraacetylatedN-azidoacetylglucosamine) for labeling O-GlcNAz-modified glycoproteins;and 3) azide-modified isoprenoid lipids, such as CLICK-IT® farnesylalcohol azide and CLICK-IT® geranylgeranyl alcohol azide. As notedabove, Applicants, have unexpectedly found that these azide-modifiedbiomolecules have anti-viral activity and can be used to treat viralinfections.

Glycosylation

Glycosylation is an enzymatic process in which carbohydrates areattached to proteins, lipids, or other organic molecules in a cell.Glycoproteins are biomolecules composed of proteins covalently linked tocarbohydrates. Certain post-translational modifications append a sugarmoiety (carbohydrate) onto a protein, thereby forming a glycoprotein.The common monosaccharides found in glycoproteins include, but are notlimited to, glucose, galactose, mannose, fucose, xylose,N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) andN-acetylneuraminic acid (NANA, also known as sialic acid).N-acetyl-D-mannosamine (ManNAc) is a precursor of the neuraminic acids,including NANA. Two of the same or different monosaccharides can jointogether to form a disaccharide. The addition of more monosaccharidesresults in the formation of oligosaccharides of increasing length. Inaddition, the sugar moiety can be a glycosyl group.

In glycoproteins, the carbohydrates can be linked to the proteincomponent by either N-glycosylation or O-glycosylation. N-glycosylationcommonly occurs through a nitrogen on an asparagine or arginine sidechain, forming an N-glycosidic linkage via an amide group.O-glycosylation commonly occurs at the hydroxy oxygen of hydroxylysine,hydroxyproline, serine, tyrosine or threonine side chains, forming anO-glycosidic linkage. GalNAc and GlcNAc are both O-linked carbohydrates.Sialic acid is found on both N- and O-linked carbohydrates.

Protein glycosylation is one of the most abundant post-translationalmodifications and plays a fundamental role in the control of biologicalsystems. For example, glycosylation influences protein folding and canhelp to stabilize proteins and prevent their degradation. Glycosylationalso can affect a protein's ability to bind to other molecules andmediate intra- or inter-cellular signaling pathways. For example,carbohydrate modifications are important for host-pathogen interactions,inflammation, development, and malignancy (Varki, A. Glycobiology 1993,3, 97-130; Lasky, L. A. Annu. Rev. Biochem. 1995, 64, 113-139. (c)Capila, I.; Linhardt, R. J. Angew. Chem., Int. Ed. 2002, 41, 391-412;Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R. A.Science 2001, 291, 2370-2376). One such covalent modification isO-GlcNAc glycosylation, which is the covalent modification of serine andthreonine residues by D-N-acetylglucosamine (Wells, L.; Vosseller, K.;Hart, G. W. Science 2001, 291, 2376-2378; Zachara, N. E.; Hart, G. W.Chem. Rev. 2002, 102, 431). The O-GlcNAc modification is found in allhigher eukaryotic organisms from C. elegans to man and has been shown tobe ubiquitous, inducible and highly dynamic, suggesting a regulatoryrole analogous to phosphorylation.

Fatty Acid Acylation

Fatty acid acylation is an enzymatic process in which fatty acids areattached to proteins in a cell. This process can affect a protein'sfunction as well as its cellular location and is common to proteins ofboth cellular and viral origin (Towler et al., Proc Natl Acad Sci USA1986, 83:2812-16). Myristic acid and palmitic acid are the two mostcommon fatty acids that are attached to proteins (Olson et al., J BiolChem 261(5):2458-66). Generally myristic acid is attached to soluble andmembrane proteins via an amide linkage to an amino terminal glycineexposed during removal of an N-methionine residue, although it can alsoattach to other amino acids. Myristoylation can also occurpost-translationally, for example, when a protease cleaves a polypeptideand exposes a glycine residue. Palmitic acid is attached to membraneproteins via an ester or thioester linkage. Myristoylation andpalmitoylation appear to play a significant role in subcellulartrafficking of proteins between membrane compartments, as well as inmodulating protein-protein interactions.

Fatty acids have two distinct regions, a long hydrophobic, hydrocarbonchain and a carboxylic acid group, which is generally ionized insolution (COO—), extremely hydrophilic and readily forms esters andamides. Natural fatty acids commonly have a chain of four to 28 carbons(usually unbranched and even numbered) and may be saturated orunsaturated. Saturated fatty acids contain no double bonds in thehydrocarbon chain and include lauric acid, myristic acid, palmitic acid,stearic acid, and arachidic acid. Unsaturated fatty acids contain atleast one double bond in the hydrocarbon chain and include myristoleicacid, palmitoleic acid, sapienic acid, oleic acid, linoleic acid,α-linoleic acid, arachidonic acid, eicosapentaenoic acid, erucic acid,and docosahexaenoic acid.

Prenylation

Protein prenylation involves the attachment of an isoprenoid lipid, suchas a farnesyl or a geranyl-geranyl moiety, to a C-terminal cysteine(s)of the target protein (McTaggert, Cell Mol Life Sci 2006, 63:255-67).These reactions are catalysed by farnesyltransferase,geranylgeranyltransferase, and Rab geranylgeranyltransferase. (Magee andSeabra, Biochem J 2003, 376:e3-4). Due to the hydrophobic nature of theisoprenoid lipid, most prenylated proteins are associated with amembrane. Most farnesylated proteins are involved in cellular signalingwhere membrane association is important for function. Isoprenoid lipidsare also important for mediating protein-protein binding throughspecialized prenyl-binding domains.

Post Translational Modifications in Viruses

Many viral proteins are extensively modified with post translationalmodifications, including, but not limited to glycosylation, acylation,and prenylation. In many instances, these post translationalmodifications are required for the virus to infect a host cell and/orevade the immune system. Post translational modifications are ofparticular importance in virology because, in general, viral genomes aresmall and thus there is heightened pressure for coding frugality. Bytaking advantage of a host's post translational machinery, viruses canexploit multiple pathways and function with minimal genomes, as a singlepost translational modification can alter a protein's function orcellular location.

For example, in HIV and Simian Immunodeficiency Viruses (SIV),glycosylation plays an important role during multiple stages of theinfectivity cycle. During infection, viral glycoproteins influence thebinding of viral proteins gp120 and gp41 to host cell CD4 receptor andCXCR4 and CCR5 co-receptors (Chen et al., Virus Res 2001, 79:91-101).Glycosylation is responsible for the proper folding and processing ofgp160 (the precursor to gp120 and gp41 (Land et al., Biochimie 2001, 83:783-90) and can enhance the interactions of HIV and SIV with differentcell types, including dendritic cells (Geijtenbeek et al., Curr TopMicrobiol Immunol 2003, 276:31-54). The normal role of gp120 in HIVbiology is to initiate viral binding to cells via CD4 receptor and CXCR4and CCR5 co-receptors expressed on the target cell. When gp120 engagesCD4, conformational changes occur in gp120 that expose co-receptorbinding sites and trigger conformational changes in gp41. Theconformational changes in gp41, in turn, expose a fusion peptide ingp41, that mediates fusion between the viral envelope and the targetcell (Chen et al., Virus Res 2001, 79:91-101). The change of onecarbohydrate at a single residue (N197) in gp120 completely changesviral tropism from CD4 tropic to CD4 independent (Kolchinksy et al., JVirol 2001, 75:3435-43). Changing the overall ratios of high mannose incomparison to complex type carbohydrates (sialic acid containing)present in gp120 affects the degree of viral binding to target cells(Fenouillet et al., J Gen Virol 1991, 1919-26). Following infection,glycosylation is required for cleavage of the envelope precursor protein(gp160) into gp120 and gp41. Upon release of the virus from an infectedcell, glycosylation is also important for immune evasion as changes inenvelope glycosylation significantly alter humoral immune responses tovirus (Kwong et al., Nature 2002, 420:678-82; Shi et al., J Gen Virol2005, 86:3385-96).

The acylation of viral proteins is also important to HIV biology. HIVbudding is a complex process involving the coordination of many cellularand viral proteins (Resh Trends Microbiol 2001, 9:57; Freed, J Virol2002, 76:4679-87). HIV budding is directed to an area of the plasmamembrane enriched in membrane rafts (Lindwasser et al., J Virol 2001,75:7913-24; Nguyen et al., J Virol 2000, 74:3264-72; Ono et al., ProcNatl Acad Sci USA 2001, 98:13925-30; Hermida-Matsumoto et al., J Virol2000, 74:8670-79), previously called lipid rafts (Pike et al., J LipidRes 2006, 47:1597-98) by myristoylation of the N-terminal glycine of thecapsid protein polyprotein precursor (pr55 gag) (Lindwasser et al., JVirol 2001, 75:7913-24; Nguyen et al., J Virol 2000, 74:3264-72; Ono etal., Proc Natl Acad Sci USA 2001, 98:13925-30). The gp120 protein isdirected to membrane rafts by palmitoylation (Yang et al., Proc NailAcad Sci USA 1995, 92:9871-75). Membrane rafts play an important role inseveral cellular processes including endocytosis, vesicle transport,cholesterol sorting, apoptosis, and signaling through the T cellreceptor (Jordan et al., J Immunol 2003, 171:78-87; Viola et al., Apmis1999, 107:615-23; Viola et al., Science 1999, 283:680-82; Bezombes etal., Curr Med Chem Anti-Canc Agents 2002, 3:263-70; Kabouridis et al.,Eur J Immunol 2000, 30:954-63). Direction of HIV proteins to theseregions may allow the virions to more efficiently hijack these pathways,thus potentially explaining the complex pathogenicity associated withdisease progression in AIDS. In fact, the removal of cholesterol, animportant membrane raft component, from HIV particles results ininactivation by at least two mechanisms, a loss of the ability to fuseto the target cell and the loss of virion integrity resulting inpermeabilization of the virus (Guyader et al, J Virol 2002, 76:10356-64;Campbell et al., J Virol 2004, 78:10556-65; Viard et al., J Virol 2002,76:11584-595; Campbell et al., Aids 2002, 16:2253-61; Liao et al, AIDSRes Hum Retroviruses 2003, 19:675-87; Graham et al., J Virol 2003,77:8237-48).

Viruses can also use the host cell machinery to modify viral proteins byadding isoprenoid lipids, such as the farnesyl and geranylgeranylgroups. For example, prenylation plays an important role in the lifecycle of the hepatitis delta virus (HDV), the etiologic agent of acuteand chronic liver disease associated with hepatitis B virus. (Einav andGlenn, J Antimicrobial Chemotherapy 2003, 52:883-86). One of the HDVproteins, the large delta antigen (LHDAg), is critical for viralassembly and undergoes farnesylation in both in vitro translationsystems and in intact cells. (Einav and Glenn, J AntimicrobialChemotherapy 2003, 52:883-86). Inhibiting prenylation by usingfarnesyltransferase inhibitors prevents HDV assembly and clears HDVviremia in a mouse model of HDV, thus underscoring the importance ofprenylation in the life cycle of certain viruses. (Einav and Glenn, JAntimicrobial Chemotherapy 2003, 52:883-86).

Similar to HIV, SIV, and HDV, other viruses rely on post translationalmodifications of viral proteins to mediate entry into host cells and/orto evade the host immune system. Thus, the azide-modified fatty acids,azide-modified carbohydrates, and azide-modified isoprenoid lipidsdescribed herein are expected to have a broad range of anti-viralactivity (such as modulating activity by inhibiting or preventingreverse transcription of the HIV viral genome, late-stage processing ofcertain viral proteins prior to final assembly of new virons, or viralentry into the cell) and can be used to treat a wide variety of viralinfections.

Methods of Use

1. Method of Treating a Viral Infection

The present disclosure provides a method of treating a plant, insect oran animal infected with a virus, the method comprising administering tothe plant, insect or animal an effective amount of an azide-modifiedfatty acid, an azide-modified carbohydrate, or an azide-modifiedisoprenoid lipid. In one embodiment, the azide-modified fatty acid is asaturated fatty acid, such as 15-azidopentadecanoic acid or12-azidododecanoic acid. In another embodiment, the azide-modifiedcarbohydrate is an N-linked carbohydrate or an O-linked carbohydrate. Inyet another embodiment, the azide-modified carbohydrate isN-azidoacetylgalactosamine, N-azidoacetyl-D-mannosamine, orN-azidoacetylglucosamine. The azide-modified carbohydrate optionallycomprises a moiety that facilitates entry into the cell including, butnot limited to, a tetraacetyl moiety. Thus, in another embodiment, theazide-modified carbohydrate is tetraacetylatedN-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine,or tetraacetylated N-azidoacetylglucosamine. In another embodiment, theisoprenoid lipid comprises a farnesyl group or a geranylgeranyl groupand includes, but is not limited to, an azido farnesyl diphosphate, anazido farnesyl alcohol, an azido geranylgeranyl diphosphate, or an azidogeranylgeranyl alcohol.

The virus may be a plant virus, an insect virus, or an animal virus. Incertain embodiments, the animal is a human and the virus is a humanvirus, such as an adenovirus, an astrovirus, a hepadnavirus, aherpesvirus, a papovavirus, a poxvirus, an arenavirus, a bunyavirus, acalcivirus, a coronavirus, a filovirus, a flavivirus, an orthomyxovirus,a paramyxovirus, a picornavirus, a reovirus, a retrovirus, arhabdovirus, or a togavirus. In one embodiment, the animal is a humanand the virus is a human immunodeficiency virus. Preferably the humanimmunodeficiency virus is HIV-1.

Whether the azide-modified fatty acid, azide-modified carbohydrate, orazide-modified isoprenoid lipid is effective to treat a viral infectioncan be determined using any of a variety of assays known in the art. Forexample, existing animal models or in vitro models of viral infectioncan be used to determine whether a given compound is effective to reduceviral load. For HIV, by way of example, the in vitro, luciferasereporter assay described in Example 2, can be used to measure theefficacy of an azide-modified compound. In a human subject, thecompound's efficacy can be determined by measuring viral load and/ormeasuring one or more symptoms of a viral infection. Viral load can bemeasured by measuring the titer or level of virus in serum. Thesemethods include, but are not limited to, a quantitative polymerase chainreaction (PCR) and a branched DNA (bDNA) test.

2. Method of Inhibiting Infectivity of a Virus

Also provided is a method of inhibiting the infectivity of a virus, themethod comprising contacting a cell infected with the virus with anazide-modified fatty acid, an azide-modified carbohydrate, or anazide-modified isoprenoid lipid in an amount effective to inhibit theinfectivity of the virus. In one embodiment, the azide-modified fattyacid is a saturated fatty acid, such as 15-azidopentadecanoic acid or12-azidododecanoic acid. In another embodiment, the azide-modifiedcarbohydrate is an N-linked carbohydrate or an O-linked carbohydrate. Inyet another embodiment, the azide-modified carbohydrate isN-azidoacetylgalactosamine, N-azidoacetyl-D-mannosamine, orN-azidoacetylglucosamine. The azide-modified carbohydrate optionallycomprises a moiety that facilitates entry into the cell including, butnot limited to, a tetraacetyl moiety. Thus, in another embodiment, theazide-modified carbohydrate is tetraacetylatedN-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine,or tetraacetylated N-azidoacetylglucosamine. In another embodiment, theisoprenoid lipid comprises a farnesyl group or a geranylgeranyl groupand includes, but is not limited to, an azido farnesyl diphosphate, anazido farnesyl alcohol, an azido geranylgeranyl diphosphate, or an azidogeranylgeranyl alcohol.

The virus may be a plant virus, an insect virus, or an animal virus. Incertain embodiments, the cell is a human cell and the virus is a humanvirus, such as an adenovirus, an astrovirus, a hepadnavirus, aherpesvirus, a papovavirus, a poxvirus, an arenavirus, a bunyavirus, acalcivirus, a coronavirus, a filovirus, a flavivirus, an orthomyxovirus,a paramyxovirus, a picornavirus, a reovirus, a retrovirus, arhabdovirus, or a togavirus. In one embodiment, the animal is a humanand the virus is a human immunodeficiency virus. Preferably the humanimmunodeficiency virus is HIV-1.

Whether the azide-modified fatty acid, azide-modified carbohydrate, orazide-modified isoprenoid lipid is effective to inhibit the infectivityof a virus can be determined using any of a variety of assays known inthe art, including a reporter gene assay, such as the luciferase assaydescribed in Example 2.

3. Method of Labeling a Viral Protein

An azide-modified fatty acid can also be used to label viral proteinsthat are modified with lipids by post-translational acylation including,but not limited to, palmitoylation and myristoylation. In suchpost-translational modifications, an azide-modified fatty acid is usedto label a viral protein. If desired, the azide labeled viral proteincan be coupled to an alkyne labeled reporter molecule using a clickchemistry reaction to permit detection of the azide labeled viralprotein.

Similarly, an azide-modified carbohydrate can be used to label viralproteins that are modified with carbohydrates by post-translationalglycosylation including, but not limited to, N-linked glycosylation andO-linked glycosylation. In such post-translational modifications, anazide-modified carbohydrate is used to label a viral protein. Ifdesired, the azide labeled viral protein can be coupled to an alkynelabeled reporter molecule using click chemistry to permit the detectionof the azide labeled viral protein.

An azide-modified isoprenoid lipid can likewise be used to label viralproteins that are modified with lipids by post-translational prenylationincluding, but not limited to, farnesylation and geranylgeranylation. Insuch post-translational modifications, an azide-modified isoprenoidlipid is used to label a viral protein. If desired, the azide labeledviral protein can be coupled to an alkyne labeled reporter moleculeusing a click chemistry reaction to permit detection of the azidelabeled viral protein.

Any virus labeled as described above may be coupled to an alkyne labeledreporter molecule or carrier molecule. The term “alkyne” includes, butis not limited to, terminal alkynes and internal alkynes, such as, forexample, cyclooctynes and difluorocyclooctynes as described by Agard etal., J. Am. Chem. Soc., 2004, 126 (46):15046-15047, dibenzocyclooctynesas described by Boon et al., WO2009/067663 A1 (2009), andaza-dibenzocyclooctynes as described by Debets et al., Chem. Comm.,2010, 46:97-99. Reporter molecules used in the methods and compositionsdescribed herein can contain, but are not limited to, a chromophore, afluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye,a nanocrystal particle, a hapten, an enzyme and a radioisotope. Incertain embodiments, such reporter molecules include fluorophores,fluorescent proteins, haptens, and enzymes.

The azide labeled virus can used to track viral infectivity in vivowhereby the virus labeled with an azide-modified carbohydrate,azide-modified fatty acid, or azide-modified isoprenoid lipid is used toinfect cultured cells or a subject. Cells can be infected over a timecourse then fixed, permeabilized, and click-labeled with a fluorescentalkyne dye. The intracellular location (or transport over a time course)of the fluorescent viral particles can be visualized, for example, bymicroscopy. Similarly, treatment of small animals with azide-labeledvirus can be performed and used to track virus bioavalability indifferent tissues including detection of the virus in circulating whiteblood cells by flow cytometry and cell/tissue section microscopy. Insome embodiments, the method of tracking a virus in vivo comprises thesteps of contacting cultured cells or a subject with an azide-modifiedcarbohydrate, azide-modified fatty acid, azide-modified isoprenoid lipidor a pharmaceutically acceptable salt thereof; contacting the culturedcells or the subject with an alkyne labeled reporter molecule; andtracking the reporter-labeled virus in the cultured cells or thesubject. In some embodiments, the method of tracking a virus in vivocomprises the steps of contacting cultured cells or a subject with anazide-modified fatty acid or pharmaceutically acceptable salt thereof;contacting the cultured cells or the subject with an alkyne labeledreporter molecule; and tracking the reporter-labeled virus in thecultured cells or the subject.

Thus, another aspect of the disclosure is directed to a method ofproducing a human immunodeficiency virus labeled with an azide-modifiedfatty acid, an azide-modified carbohydrate, or an azide-modifiedisoprenoid lipid, the method comprising contacting a cell infected withthe human immunodeficiency virus with the azide-modified fatty acid, theazide-modified carbohydrate, or the azide-modified isoprenoid lipid sothat the azide-modified fatty acid, the azide-modified carbohydrate, orthe azide-modified isoprenoid lipid enters the cell and is incorporatedinto a protein of the virus, thereby producing the labeled virus. In oneembodiment, the azide-modified fatty acid is a saturated fatty acid,such as 15-azidopentadecanoic acid or 12-azidododecanoic acid. Inanother embodiment, the azide-modified carbohydrate is an N-linkedcarbohydrate or an O-linked carbohydrate. In yet another embodiment, theazide-modified carbohydrate is N-azidoacetylgalactosamine,N-azidoacetyl-D-mannosamine, or N-azidoacetylglucosamine. In anotherembodiment, the isoprenoid lipid comprises a farnesyl group or ageranylgeranyl group and includes, but is not limited to, an azidofarnesyl diphosphate, an azido farnesyl alcohol, an azido geranylgeranyldiphosphate, or an azido geranylgeranyl alcohol. The azide-modifiedcarbohydrate optionally comprises a moiety that facilitates entry intothe cell including, but not limited to, a tetraacetyl moiety. Thus, inanother embodiment, the azide-modified carbohydrate is tetraacetylatedN-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine,or tetraacetylated N-azidoacetylglucosamine. In certain embodiments, thecell is a human cell.

Viruses

The azide-modified fatty acids, azide-modified carbohydrates, orazide-modified isoprenoid lipids preferably target post translationalmodifications common to most viruses and thus represent a new class ofanti-viral agents with potential for anti-viral activity against a broadspectrum of viruses. In principle, these compounds may be used to treata plant, insect or an animal infected with any virus. In someembodiments, the virus is a plant virus. In some embodiments, the virusis an insect virus. In other embodiments, the virus is an animal virus.In yet other embodiments, the virus is a human virus. In one embodiment,the virus is one that infects a non-human mammal, such as a mammalianlivestock animal, including, but not limited to, a cow, a horse, a pig,a goat, or a sheep.

In other embodiments, the virus is a DNA virus. DNA viruses include, butare not limited to a virus belonging to one of the following families:adenovirus, astrovirus, hepadnavirus, herpesvirus, papovavirus, andpoxvirus. In other embodiments, the virus is an RNA virus. RNA virusesinclude but are not limited to a virus belonging to one the followingfamilies: arenavirus, bunyavirus, calcivirus, coronavirus, filovirus,flavivirus, orthomyxovirus, paramyxovirus, picornavirus, reovirus,retrovirus, rhabdovirus, and togavirus.

1. Non-Human Animal Viruses

In methods directed to treating a viral infection or inhibiting viralinfectivity in a non-human animal, the animal virus is preferablyselected from a picornavirus, such as a bovine enterovirus, a porcineenterovrus B, a foot-and-mouth disease virus, an equine rhinitis Avirus, a bovine rhinitis B virus, a ljungan virus, equine rhinitis Bvirus, an aichi virus, a bovine kobuvirus, a porcine teschovirus, aporcine sapelovirus, a simian sapelovirus, an avian sapelovirus, anavian encephalomyelitis virus, a duck hepatitis A virus, or a simianenterovirus A; a pestivirus, such as border disease virus, a bovinevirus diarrhea, or a classical swine fever virus; an arterivirus, suchas an equine arteritis virus, a porcine reproductive and respiratorysyndrome virus, a lactate dehydrogenase elevating virus, or a simianhaemorrhagic fever virus; a coronavirus, such as a bovine coronavirus, aporcine coronavirus, a feline coronavirus, or a canine coronavirus; aparamyxovirus, such as a hendra virus, a nipah virus, a canine distempervirus, a rinderpest virus, a Newcastle disease virus, and a bovinerespiratory syncytial virus; an orthomyxovirus, such as an influenza Avirus, an influenza B virus, or an influenza C virus; a reovirus, suchas a bluetongue virus; a porcine circovirus, a herpesvirus, such as apseudorabies virus or a bovine herpesvirus 1; an asfarvirus, such as anAfrican swine fever virus; a retrovirus, such as a simianimmunodeficiency virus, a feline immunodeficiency virus, a bovineimmunodeficiency virus, a bovine leukemia virus, a feline leukemiavirus, a Jaagsiekte sheep retrovirus, or a caprine arthritisencephalitis virus; a flavivirus, such as a yellow fever virus, a WestNile virus, a dengue fever virus, a tick borne encephalitis virus, or abovine viral diarrhea; or a rhabdovirus, such as a rabies virus.

2. Human Animal Viruses

In methods directed to treating a viral infection or inhibiting viralinfectivity in a human, the human virus is preferably selected from anadenovirus, an astrovirus, a hepadnavirus, a herpesvirus, a papovavirus,a poxvirus, an arenavirus, a bunyavirus, a calcivirus, a coronavirus, afilovirus, a flavivirus, an orthomyxovirus, a paramyxovirus, apicornavirus, a reovirus, a retrovirus, a rhabdovirus, or a togavirus.

In preferred embodiments, the adenovirus includes, but is not limitedto, a human adenovirus. In preferred embodiments, the astrovirusincludes, but is not limited to, a mamastrovirus. In preferredembodiments, the hepadnavirus includes, but is not limited to, thehepatitis B virus. In preferred embodiments, the herpesvirus includes,but is not limited to, a herpes simplex virus type I, a herpes simplexvirus type 2, a human cytomegalovirus, an Epstein-Barr virus, avaricella zoster virus, a roseolovirus, and a Kaposi'ssarcoma-associated herpesvirus. In preferred embodiments, thepapovavirus includes, but is not limited to, human papilloma virus and ahuman polyoma virus. In preferred embodiments, the poxvirus includes,but is not limited to, a variola virus, a vaccinia virus, a cowpoxvirus, a monkeypox virus, a smallpox virus, a pseudocowpox virus, apapular stomatitis virus, a tanapox virus, a yaba monkey tumor virus,and a molluscum contagiosum virus. In preferred embodiments, thearenavirus includes, but is not limited to lymphocytic choriomeningitisvirus, a lassa virus, a machupo virus, and a junin virus. In preferredembodiments, the bunyavirus includes, but is not limited to, a hantavirus, a nairovirus, an orthobunyavirus, and a phlebovirus. In preferredembodiments, the calcivirus includes, but is not limited to, avesivirus, a norovirus, such as the Norwalk virus and a sapovirus. Inpreferred embodiments, the coronavirus includes, but is not limited to,a human coronavirus (etiologic agent of severe acute respiratorysyndrome (SARS)). In preferred embodiments, the filovirus includes, butis not limited to, an Ebola virus and a Marburg virus. In preferredembodiments, the flavivirus includes, but is not limited to, a yellowfever virus, a West Nile virus, a dengue fever virus, a hepatitis Cvirus, a tick borne encephalitis virus, a Japanese encephalitis virus, aMurray Valley encephalitis virus, a St. Louis encephalitis virus, aRussian spring-summer encephalitis virus, a Omsk hemorrhagic fevervirus, a bovine viral diarrhea virus, a Kyasanus Forest disease virus,and a Powassan encephalitis virus. In preferred embodiments, theorthomyxovirus includes, but is not limited to, influenza virus type A,influenza virus type B, and influenza virus type C. In preferredembodiments, the paramyxovirus includes, but is not limited to, aparainfluenza virus, a rubula virus (mumps), a morbillivirus (measles),a pneumovirus, such as a human respiratory syncytial virus, and asubacute sclerosing panencephalitis virus. In preferred embodiments, thepicornavirus includes, but is not limited to, a poliovirus, arhinovirus, a coxsackievirus A, a coxsackievirus B, a hepatitis A virus,an echovirus, and an eneterovirus. In preferred embodiments, thereovirus includes, but is not limited to, a Colorado tick fever virusand a rotavirus. In preferred embodiments, the retrovirus includes, butis not limited to, a lentivirus, such as a human immunodeficiency virus,and a human T-lymphotrophic virus (HTLV). In preferred embodiments, therhabdovirus includes, but is not limited to, a lyssavirus, such as therabies virus, the vesicular stomatitis virus and the infectioushematopoietic necrosis virus. In preferred embodiments, the togavirusincludes, but is not limited to, an alphavirus, such as a Ross rivervirus, an O'nyong'nyong virus, a Sindbis virus, a Venezuelan equineencephalitis virus, an Eastern equine encephalitis virus, and a Westernequine encephalitis virus, and a rubella virus.

3. Plant Viruses

In methods directed to treating a viral infection or inhibiting viralinfectivity in a plant, the plant virus is selected from an alfamovirus,an allexivirus, an alphacryptovirus, an anulavirus, an apscaviroid, anaureusvirus, an avenavirus, an aysunviroid, a badnavirus, a begomovirus,a benyvirus, a betacryptovirus, a betaflexiviridae, a bromovirus, abymovirus, a capillovirus, a carlavirus, a carmovirus, a caulimovirus, acavemovirus, a cheravirus, a closterovirus, a cocadviroid, a coleviroid,a comovirus, a crinivirus, a cucumovirus, a curtovirus, acytorhabdovirus, a dianthovirus, an enamovirus, an umbravirus & B-typesatellite virus, a fabavirus, a fijivirus, a furovirus, a hordeivirus, ahostuviroid, an idaeovirus, an ilarvirus, an ipomovirus, a luteovirus, amachlomovirus, a macluravirus, a marafivirus, a mastrevirus, ananovirus, a necrovirus, a nepovirus, a nucleorhabdovirus, an oleavirus,an ophiovirus, an oryzavirus, a panicovirus, a pecluvirus, a petuvirus,a phytoreovirus, a polerovirus, a pomovirus, a pospiviroid, apotexvirus, a potyvirus, a reovirus, a rhabdovirus, a rymovirus, asadwavirus, a SbCMV-like virus, a sequivirus, a sobemovirus, atenuivirus, a TNsatV-like satellite virus, a tobamovirus, a topocuvirus,a tospovirus, a trichovirus, a tritimovirus, a tungrovirus, a tymovirus,an umbravirus, a varicosavirus, a vitivirus, or a waikavirus.

4. Insect Viruses

In methods directed to labeling an insect virus, treating an insectvirus infection, or inhibiting insect viral infectivity, the insectvirus is preferably selected from a densovirus, such as Junonia coeniadensovirus, Bombyx mori densovirus, Aedes aegypti densovirus, orPeriplanta fuliginosa densovirus; an iridovirus, such as iridescentvirus 6; a chloriridovirus, a baculovirus, such as nuclear polyhedrosisvirus or a granulovirus; a polydnavirus, such as a ichnovirus or abracovirus; an entomopox virus, such as an entomopox A virus, anentomopox B virus, or an entomopox C virus; an ascovirus, such as aSpodoptera frugiperda ascovirus 1a, a Trichoplusia ni ascovirus 2a, or aDiadromus pulchellus ascovirus 4a; an insect picornavirus, such as a beeacute paralysis virus, a Drosophila P, C, or A virus, a bee virus Xvirus, or a silkworm flacherie virus; a calicivirus; a nodavirus, suchas a black beetle virus, a flock house virus, a nodamura virus, apariacoto virus, or a gypsy moth virus.

Azide-Modified Biomolecules

The azide-modified biomolecules described herein represent a new classof anti-viral agents. In certain embodiments, the azide-modifiedbiomolecule is a carbohydrate or a pharmaceutically acceptablederivative or prodrug thereof. The carbohydrate can be selected from awide variety of carbohydrates commercially available and/or widely knownto those skilled in the art. In preferred embodiments, the carbohydrateis selected to prevent, inhibit and/or retard viral infection of cells.Preferably the carbohydrate is naturally occurring. It is appreciatedthat the azide-containing carbohydrate, whether naturally occurring ornot, may be modified, for example, by short chain alkylation such asmethylation or acetylation, esterification, as well as otherderivatizations that maintain antiviral activity.

In one embodiment, the carbohydrate is one that is attached directly orindirectly to a protein through a glycosylation reaction in a cell. Inone embodiment, the carbohydrate is an N-linked carbohydrate or anO-linked carbohydrate. In yet another embodiment, the carbohydrate isN-azidoacetylgalactosamine, N-azidoacetyl-D-mannosamine, orN-azidoacetylglucosamine.

In certain embodiments, the azide-modified carbohydrate contains amoiety that facilitates entry into the cell including, but not limitedto, a tetraacetyl moiety. Thus, in one embodiment, the azide-modifiedcarbohydrate is a tetraacetylated version of an N-linked carbohydrate oran O-linked carbohydrate. In yet another embodiment, the azide-modifiedcarbohydrate is tetraacetylated N-azidoacetylgalactosamine,tetraacetylated N-azidoacetyl-D-mannosamine, or tetraacetylatedN-azidoacetylglucosamine.

In other embodiments, the azide-modified biomolecule is a fatty acid ora pharmaceutically acceptable derivative or prodrug thereof. The fattyacid can be selected from a wide variety of fatty acids commerciallyavailable and/or widely known to those skilled in the art. In preferredembodiments, the fatty acid is selected to prevent, inhibit and/orretard viral infection of cells. Preferably, the fatty acid is naturallyoccurring.

In one embodiment, the fatty acid is saturated or unsaturated and has ahydrocarbon chain with an even number of carbon atoms, such as 4-24carbon atoms. Suitable unsaturated free fatty acids have a hydrocarbonchain with 14-24 carbon atoms and include palmitoleic acid, oleic acid,linoleic acid, alpha and gamma linolenic acid, arachidonic acid,eicosapentanoic acid and tetracosenoic acid. Suitable saturated fattyacids have a hydrocarbon chain with 4-18 carbon atoms and are preferablyselected from butyric or isobutyric acid, succinic acid, caproic acid,adipic acid, caprylic acid, capric acid, lauric acid, myristic acid,palmitic acid stearic acid, and arachidic acid. It is appreciated thatthe azide-containing fatty acid, whether naturally occurring or not, maybe modified by chemical substitution including, but not limited to,short chain alkylation such as methylation or acetylation,esterification, as well as other derivitisations that maintain antiviralactivity. In addition, it is possible to replace the fatty acid in theazide-modified biomolecule with an alkyne, ketone, or other smallmolecule that has been shown to be metabolically compatible.

In some embodiments, azide-modified fatty acid or pharmaceuticallyacceptable salt thereof, has the formula: Y—CH₂—X—CO₂H, wherein, Y is Hor an azido group; and when Y is an azido group, X is a linear orbranched carbon chain comprising 6 to 28 carbons, wherein one or more ofsaid carbons may be independently replaced by an oxygen, selenium,silicon, sulfur, SO, SO₂ or NR₁, or wherein one or more pairs of saidcarbons adjacent to one another may be attached to one another by adouble or triple bond; or when Y is H, X is a linear or branched carbonchain comprising 6 to 28 carbons, wherein one hydrogen on one of saidcarbons is replaced with an azido group and wherein one or more of saidcarbons not having an the azido group attached thereto may beindependently replaced by an oxygen, selenium, silicon, sulfur, SO, SO₂or NR₁, or wherein one or more pairs of said carbons adjacent to oneanother and not having an azido group may be attached to one another bya double or triple bond; wherein, R₁ is H or an alkyl comprising 1 to 6carbons.

In one embodiment, the fatty acid is one that is attached to a proteinthrough an acylation reaction (e.g., palmitoylation or myristoylation)in a cell. Thus, in one embodiment, the azide-modified fatty acid is asaturated fatty acid, such as 15-azidopentadecanoic acid (palmitic acid,azide) or 12-azidododecanoic acid (myristic acid, azide). The compoundsused in the methods of the present invention may be present in the formof pharmaceutically acceptable salts. For use in medicines, the salts ofthe compounds of the invention refer to non-toxic pharmaceuticallyacceptable salts.

The pharmaceutically acceptable salts of these compounds include acidaddition salts and base addition salts. The term “pharmaceuticallyacceptable salts” embraces salts commonly used to form alkali metalsalts and to form addition salts of free acids or free bases. The natureof the salt is not critical, provided that it is pharmaceuticallyacceptable.

Suitable pharmaceutically acceptable acid addition salts of thesecompounds may be prepared from an inorganic acid or an organic acid.Examples of such inorganic acids are hydrochloric, hydrobromic,hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriateorganic acids may be selected from aliphatic, cycloaliphatic, aromatic,arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organicacids, examples of which are formic, acetic, propionic, succinic,glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic,toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic,algenic, β-hydroxybutyric, malonic, galactic, and galacturonic acid.

Pharmaceutically acceptable acidic/anionic salts also include, theacetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide,calcium edetate, camsylate, carbonate, chloride, citrate,dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, malonate, mandelate, mesylate,methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate,phosphate/diphospate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, hydrogensulfate, tannate, tartrate,teoclate, tosylate, and triethiodide salts.

Suitable pharmaceutically acceptable base addition salts of thedisclosed compounds include, but are not limited to, metallic salts madefrom aluminum, calcium, lithium, magnesium, potassium, sodium and zincor organic salts made from N,N′-dibenzylethylene-diamine,chloroprocaine, choline, diethanolamine, ethylenediamine,N-methylglucamine, lysine, arginine and procaine. All of these salts maybe prepared by conventional means from the corresponding compoundrepresented by the disclosed compound by treating, for example, thedisclosed compounds with the appropriate acid or base. Pharmaceuticallyacceptable basic/cationic salts also include, the diethanolamine,ammonium, ethanolamine, piperazine and triethanolamine salts.

Such salts can be formed as known to those skilled in the art.

In yet another embodiment, the azide-modified biomolecule is anazide-modified isoprenoid lipid or a pharmaceutically acceptablederivative or prodrug thereof. The isoprenoid lipid can be selected froma wide variety of isoprenoid lipids commercially available and/or widelyknown to those skilled in the art. In preferred embodiments, theisoprenoid lipid is selected to prevent, inhibit and/or retard viralinfection of cells. Preferably, the isoprenoid lipid is naturallyoccurring. It is appreciated that the azide-containing isoprenoid lipid,whether naturally occurring or not, may be modified, for example, byshort chain alkylation such as methylation or acetylation,esterification, as well as other derivatizations that maintain antiviralactivity.

In one embodiment, the isoprenoid lipid is one that is attached to aprotein through a prenylation reaction in a cell. In one embodiment, theisoprenoid lipid, in the presence of the catalytic activity of afarnesyltransferase or a geranylgeranyltransferase, is attached to aprotein in a cell. In another embodiment, the isoprenoid lipid comprisesa farnesyl group or a geranylgeranyl group and includes, but is notlimited to, an azido farnesyl diphosphate, an azido farnesyl alcohol, anazido geranylgeranyl diphosphate, or an azido geranylgeranyl alcohol.The azide-modified carbohydrates, azide-modified fatty acids, andazide-modified isoprenoid lipids described herein can be prepared usingmethods known in the art, including those disclosed in U.S. PatentApplication Publication No. 20050222427, U.S. Patent ApplicationPublication No. 2007/0249014, and Hang, H. C. et al., J Am Chem Soc2007, 129:2744-45, the disclosures of which are incorporated byreference in their entirety.

Combination Therapy

In one embodiment, a pharmaceutical composition comprising anazide-modified fatty acid, an azide-modified carbohydrate, or anazide-modified isoprenoid lipid and at least one anti-viral agent isadministered in combination therapy. The therapy is useful for treatingviral infections, including, but not limited to, an HIV infection. Theterm “in combination” in this context means that the azide-modifiedfatty acid, azide-modified carbohydrate, or the azide-modifiedisoprenoid lipid and the anti-viral agent are given substantiallycontemporaneously, either simultaneously or sequentially. In oneembodiment, if given sequentially, at the onset of administration of thesecond compound, the first of the two compounds is still detectable ateffective concentrations at the site of treatment. In anotherembodiment, if given sequentially, at the onset of administration of thesecond compound, the first of the two compounds is not detectable ateffective concentrations at the site of treatment.

For example, the combination therapy can include an azide-modified fattyacid, an azide-modified carbohydrate, or an azide-modified isoprenoidlipid co-formulated with, and/or co-administered with, at least oneadditional anti-viral agent. Although specific examples of anti-viralagents are provided, in principle, the azide-modified fatty acid,azide-modified carbohydrate, or an azide-modified isoprenoid lipid canbe combined with any pharmaceutical composition useful for treating aviral infection. Such combination therapies may advantageously use lowerdosages of the administered therapeutic agents, thus avoiding possibletoxicities or complications associated with the various monotherapies.Moreover, the additional anti-viral agents disclosed herein act onpathways or stage of viral infection in addition to or that differ fromthe pathway stage of viral infection affected by the azide-modifiedfatty acid, the azide-modified carbohydrate, or the azide-modifiedisoprenoid lipid, and thus are expected to enhance and/or synergize withthe effects of the azide-modified fatty acid, the azide-modifiedcarbohydrate, or the azide-modified isoprenoid lipid. The additionalanti-viral agent may include at least one reverse transcriptaseinhibitor, a virus protease inhibitor, a viral fusion inhibitor, a viralintegrase inhibitor, a glycosidase inhibitor, a viral neuraminidaseinhibitor, an M2 protein inhibitor, an amphotericin B, hydroxyurea,α-interferon, β-interferon, γ-interferon, and an antisenseoligonucleotide.

The at least one reverse transcriptase inhibitor includes, but is notlimited to, one or more nucleoside analogs, such as Zidovudine (AZT),Didanosine (ddI), Zalcitabine (ddC), Stavudine (d4T), Lamivudine (3TC),Abacavir (ABC), Emtricitabine (FTC), Entecavir (INN), Apricitabine(ATC), Atevirapine, ribavirin, acyclovir, famciclovir, valacyclovir,ganciclovir, and valganciclovir; one or more nucleotide analogs, such asTenofovir (tenofovir disoproxil fumarate), Adefovir (bis-POM PMPA),PMPA, and cidofovir; or one or more non-nucleoside reverse transcriptaseinhibitors, such as Efavirenz, Nevirapine, Delavirdine, and Etravirine.

The at least one viral protease inhibitor includes, but is not limitedto, tipranavir, darunavir, indinavir, lopinavir, fosamprenavir,atazanavir, saquinavir, ritonavir, indinavir, nelfinavir, andamprenavir.

The at least one viral fusion inhibitor includes, but is not limited toa CD4 antagonist, such as soluble CD4 or an antibody that binds to CD4,such as TNX-355, BMS-806; a CCR5 antagonist, such as SCH—C, SCH-D,UK-427,857, maraviroc, vicriviroc, or an antibody that binds to CCR5,such as PRO-140; a CXCR4 antagonist, such as, AMD3100 or AMD070; or anantagonist of gp41, such as enfuvirtide.

The at least one viral integrase inhibitor includes, but is not limitedto, raltegravir.

The at least one glycosidase inhibitor includes, but is not limited to,SC-48334 or MDL-28574.

The at least one viral neuraminidase inhibitor includes, but is notlimited to, oseltamivir, peramivir, zanamivir, and laninamivir.Neuraminidase is a protein on the surface of influenza viruses thatmediates the virus' release from an infected cell. (Bossart-Whitaker etal., J Mol Biol, 1993, 232:1069-83). The influenza virus attaches to thecell membrane using the viral hemagglutinin protein. The hemagglutininprotein binds to sialic acid moieties found on glycoproteins in the hostcell's membranes. In order for the virus to be released from the cell,neuraminidase must enzymatically cleave the sialic acid groups from thehost glycoproteins. Thus, inhibiting neuraminidase prevents the releaseof the influenza virus from an infected cell.

The at least one M2 inhibitor includes, but is not limited to,amantadine and rimantidine. M2 is an ion channel protein found in theviral envelope of the influenza virus (Henckel et al., J Biol Chem,1998, 273:6518-24). The M2 protein plays an important role incontrolling the uncoating of the influenza virus, leading to the releaseof the virion contents into the host cell cytoplasm. Blocking M2inhibits viral replication.

An azide-modified fatty acid, an azide-modified carbohydrate, or anazide-modified isoprenoid lipid disclosed herein can be used incombination with other therapeutic agents to treat specific viralinfections as discussed in further detail below.

Non-limiting examples of agents for treating an HIV infection, withwhich the azide-modified fatty acid, the azide-modified carbohydrate, orthe azide-modified isoprenoid lipid can be combined include at least oneof the following: Zidovudine (AZT), Didanosine (ddI), Zalcitabine (ddC),Stavudine (d4T), Lamivudine (3TC), Abacavir (ABC), Emtricitabine (FTC),Entecavir (INN), Apricitabine (ATC), Tenofovir (tenofovir disoproxilfumarate), Adefovir (bis-POM PMPA) Efavirenz, Nevirapine, Delavirdine,Etravirine, tipranavir, darunavir, indinavir, lopinavir, fosamprenavir,atazanavir, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, aCD4 antagonist, such as soluble CD4 or an antibody that binds to CD4,such as TNX-355, BMS-806, a CCR5 antagonist, such as SCH-C, SCH-D,UK-427,857, maraviroc, vicriviroc, or an antibody that binds to CCR5,such as PRO-140, a CXCR4 antagonist, such as, AMD3100 or AMD070, or anantagonist of gp41, such as enfuvirtide.

Specific examples of combination therapy that can be used to treat HIVinfection include, but are not limited to, an azide-modified fatty acid,azide-modified carbohydrate, or the azide-modified isoprenoid lipidcombined with: 1) tenofovir, emtricitabine, and efavirenz; 2) lopinavirand ritonavir; 3) lamivudine and zidovudine; 4) abacavir, lamivudine,and zidovudine; 5) lamivudine and abacavir; or 6) tenofovir andemtricitabine.

Non-limiting examples of agents for a herpesvirus infection with whichthe azide-modified fatty acid, the azide-modified carbohydrate, or theazide-modified isoprenoid lipid can be combined include acyclovir,famciclovir, valacyclovir, cidofovir, foscarnet, ganciclovir, andvalganciclovir.

Non-limiting examples of agents for an influeneza virus infection withwhich the azide-modified fatty acid, the azide-modified carbohydrate, orthe azide-modified isoprenoid lipid can be combined include amantadine,rimantidine, oseltamivir, peramivir, zanamivir, and laninamivir.

Non-limiting examples of agents for a respiratory synctial virusinfection with which the azide-modified fatty acid, the azide-modifiedcarbohydrate, or the azide-modified isoprenoid lipid can be combinedinclude ribavirin.

Another aspect of the present invention accordingly relates to kits forcarrying out the combined administration of the azide-modified fattyacid, the azide-modified carbohydrate, or the azide-modified isoprenoidlipid with other therapeutic agents. In one embodiment, the kitcomprises the azide-modified fatty acid, the azide-modifiedcarbohydrate, or the azide-modified isoprenoid lipid formulated in apharmaceutical excipient, and at least one anti-viral agent, formulatedas appropriate in one or more separate pharmaceutical preparations.

Pharmaceutical Compositions and Methods of Administration

This disclosure provides compositions that are suitable forpharmaceutical use and administration to patients. The pharmaceuticalcompositions comprise an azide-modified fatty acid, an azide-modifiedcarbohydrate, an azide-modified isoprenoid lipid or any of the compoundsherein and a pharmaceutically acceptable excipient. In one embodiment,the azide-modified fatty acid is a saturated fatty acid, such as15-azidopentadecanoic acid or 12-azidododecanoic acid. In anotherembodiment, the azide-modified carbohydrate is an N-linked carbohydrateor an O-linked carbohydrate. In yet another embodiment, theazide-modified carbohydrate is N-azidoacetylgalactosamine,N-azidoacetyl-D-mannosamine, or N-azidoacetylglucosamine. In anotherembodiment, the azide-modified carbohydrate contains a moiety thatfacilitates entry into the cell including, but not limited to, atetraacetyl moiety. Thus, in one embodiment, the azide-modifiedcarbohydrate is a tetraacetylated version of an N-linked carbohydrate oran O-linked carbohydrate. In yet another embodiment, the azide-modifiedcarbohydrate is tetraacetylated N-azidoacetylgalactosamine,tetraacetylated N-azidoacetyl-D-mannosamine, or tetraacetylatedN-azidoacetylglucosamine. In another embodiment, the isoprenoid lipidcomprises a farnesyl or a geranylgeranyl group and includes, but is notlimited to, an azido farnesyl diphosphate, an azido farnesyl alcohol, anazido geranylgeranyl diphosphate, or an azido geranylgeranyl alcohol.The pharmaceutical compositions may also be included in a container,pack, or dispenser together with instructions for administration.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Methods to accomplish theadministration are known to those of ordinary skill in the art.Pharmaceutical compositions may be topically or orally administered, orcapable of transmission across mucous membranes. Examples ofadministration of a pharmaceutical composition include oral ingestion orinhalation. Administration may also be intravenous, intraperitoneal,intramuscular, intracavity, subcutaneous, cutaneous, or transdermal.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include at least one of the following components:a sterile diluent such as water, saline solution, fixed oils,polyethylene glycol, glycerine, propylene glycol, or other syntheticsolvent; antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate,citrate, or phosphate; and tonicity agents such as sodium chloride ordextrose. The pH can be adjusted with acids or bases. Such preparationsmay be enclosed in ampoules, disposable syringes, or multiple dosevials.

Solutions or suspensions used for intravenous administration include acarrier such as physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.), ethanol, or polyol. In all cases, thecomposition must be sterile and fluid for easy syringability. Properfluidity can often be obtained using lecithin or surfactants. Thecomposition must also be stable under the conditions of manufacture andstorage. Prevention of microorganisms can be achieved with antibacterialand antifungal agents, e.g., parabens, chlorobutanol, phenol, ascorbicacid, thimerosal, etc. In many cases, isotonic agents (sugar),polyalcohols (mannitol and sorbitol), or sodium chloride may be includedin the composition. Prolonged absorption of the composition can beaccomplished by adding an agent which delays absorption, e.g., aluminummonostearate and gelatin.

Oral compositions include an inert diluent or edible carrier. Thecomposition can be enclosed in gelatin or compressed into tablets. Forthe purpose of oral administration, the azide-modified fatty acid, theazide-modified carbohydrate, or the azide-modified isoprenoid lipid canbe incorporated with excipients and placed in tablets, troches, orcapsules. Pharmaceutically compatible binding agents or adjuvantmaterials can be included in the composition. The tablets, troches, andcapsules, may contain (1) a binder such as microcrystalline cellulose,gum tragacanth or gelatin; (2) an excipient such as starch or lactose,(3) a disintegrating agent such as alginic acid, Primogel, or cornstarch; (4) a lubricant such as magnesium stearate; (5) a glidant suchas colloidal silicon dioxide; or (6) a sweetening agent or a flavoringagent.

The composition may also be administered by a transmucosal ortransdermal route. Transmucosal administration can be accomplishedthrough the use of lozenges, nasal sprays, inhalers, or suppositories.Transdermal administration can also be accomplished through the use of acomposition containing ointments, salves, gels, or creams known in theart. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used.

For administration by inhalation, the azide-modified fatty acid, theazide-modified carbohydrate, or the azide-modified isoprenoid lipid aredelivered in an aerosol spray from a pressured container or dispenser,which contains a propellant (e.g., liquid or gas) or a nebulizer. Incertain embodiments, the azide-modified fatty acid, the azide-modifiedcarbohydrate, or the azide-modified isoprenoid lipid is prepared with acarrier to protect the compounds against rapid elimination from thebody. Biodegradable polymers (e.g., ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylacticacid) are often used. Methods for the preparation of such formulationsare known by those skilled in the art.

In other embodiments, the composition comprises a delivery agent fordelivering the azide-modified fatty acid, the azide-modifiedcarbohydrate, or the azide-modified isoprenoid lipid to a cell includingbut not limited to, a liposome. Liposomes (also known as lipid vesicles)are colloidal particles that are prepared from polar lipid moleculesderived either from natural sources or chemical synthesis. Suchspherical, closed structures composed of curved lipid bilayers, aretypically used to entrap drugs, which are often cytotoxic, in order toreduce toxicity and/or increase efficacy. Liposome-entrapped drugpreparations are often provided in a dry (e.g. freeze-dried) form, whichis subsequently reconstituted with an aqueous solution immediately priorto administration. This is done in order to minimize the possibility ofleakage of e.g. cytotoxic drug into aqueous solution and therebyreducing the entrapping effect of the liposome.

Examples of formulations comprising inter alia liposome-encapsulatedactive ingredients are discussed in U.S. Pat. No. 4,427,649, U.S. Pat.No. 4,522,811, U.S. Pat. No. 4,839,175, U.S. Pat. No. 5,569,464, EP 249561, WO 00/38681, WO 88/01862, WO 98/58629, WO 98/00111, WO 03/105805,U.S. Pat. No. 5,049,388, U.S. Pat. No. 5,141,674, U.S. Pat. No.5,498,420, U.S. Pat. No. 5,422,120, WO 87/01586, WO 2005/039533, US2005/0112199 and U.S. Pat. No. 6,228,393, all of which are herebyincorporated by reference in their entirety.

The azide-modified fatty acid, azide-modified carbohydrate, orazide-modified isoprenoid lipid containing compositions are administeredin therapeutically effective amounts as described. Therapeuticallyeffective amounts may vary with the subject's age, condition, sex, andseverity of medical condition. Appropriate dosage may be determined by aphysician based on clinical indications. The azide-modified fatty acid,azide-modified carbohydrate, or azide-modified isoprenoid lipidcontaining composition may be given as a bolus dose to maximize thecirculating levels of the azide-modified fatty acid, azide-modifiedcarbohydrate, or the azide-modified isoprenoid lipid for the greatestlength of time. Continuous infusion may also be used after the bolusdose.

Examples of dosage ranges that can be administered to a subject can bechosen from: 1 μg/kg to 20 mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to 1mg/kg, 10 μg/kg to 1 mg/kg, 10 μg/kg to 100 μg/kg, 100 μg/kg to 1 mg/kg,250 μg/kg to 2 mg/kg, 250 μg/kg to 1 mg/kg, 500 μg/kg to 2 mg/kg, 500μg/kg to 1 mg/kg, 1 mg/kg to 2 mg/kg, 1 mg/kg to 5 mg/kg, 5 mg/kg to 10mg/kg, 10 mg/kg to 20 mg/kg, 15 mg/kg to 20 mg/kg, 10 mg/kg to 25 mg/kg,15 mg/kg to 25 mg/kg, 20 mg/kg to 25 mg/kg, and 20 mg/kg to 30 mg/kg (orhigher). These dosages may be administered daily, weekly, biweekly,monthly, or less frequently, for example, biannually, depending ondosage, method of administration, disorder or symptom(s) to be treated,and individual subject characteristics. Dosages can also be administeredvia continuous infusion (such as through a pump). The administered dosemay also depend on the route of administration. For example,subcutaneous administration may require a higher dosage than intravenousadministration.

In certain circumstances, it may be advantageous to formulatecompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited for the patient. Each dosage unitcontains a predetermined quantity of azide-modified fatty acid, theazide-modified carbohydrate, or the azide-modified isoprenoid lipidcalculated to produce a therapeutic effect in association with thecarrier. The dosage unit depends on the characteristics of theazide-modified fatty acid, the azide-modified carbohydrate, or theazide-modified isoprenoid lipid and the particular therapeutic effect tobe achieved.

Toxicity and therapeutic efficacy of the composition can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.

The data obtained from the cell culture assays and animal studies can beused to formulate a dosage range in humans. The dosage of thesecompounds may lie within the range of circulating concentrations of theazide-modified fatty acid, azide-modified carbohydrate, orazide-modified isoprenoid lipid in the blood, that includes an ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage composition form employed and the route ofadministration. For any azide-modified fatty acid, azide-modifiedcarbohydrate, or azide-modified isoprenoid lipid used in the methodsdescribed herein, the therapeutically effective dose can be estimatedinitially using cell culture assays. A dose may be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC₅₀ (i.e., the concentration of antibody which achieves ahalf-maximal inhibition of symptoms). The effects of any particulardosage can be monitored by a suitable bioassay.

The compositions may also contain other active compounds providingsupplemental, additional, or enhanced therapeutic functions. In oneembodiment, the composition further comprises at least one anti-viralagent, such as a reverse transcriptase inhibitor, a virus proteaseinhibitor, a viral fusion inhibitor, a viral integrase inhibitor, aglycosidase inhibitor, an amphotericin B, hydroxyurea, α-interferon,β-interferon, γ-interferon, and an antisense oligonucleotide.

In one embodiment, the at least one reverse transcriptase inhibitorincludes, but is not limited to, one or more nucleoside analogs, such asZidovudine (AZT), Didanosine (ddI), Zalcitabine (ddC), Stavudine (d4T),Lamivudine (3TC), Abacavir (ABC), Emtricitabine (FTC), Entecavir (INN),Apricitabine (ATC), Atevirapine, ribavirin, acyclovir, famciclovir,valacyclovir, ganciclovir, and valganciclovir; one or more nucleotideanalogs, such as Tenofovir (tenofovir disoproxil fumarate), Adefovir(bis-POM PMPA), PMPA, and cidofovir; or one or more non-nucleosidereverse transcriptase inhibitors, such as Efavirenz, Nevirapine,Delavirdine, and Etravirine.

In other embodiments, the at least one viral protease inhibitorincludes, but is not limited to, tipranavir, darunavir, indinavir,lopinavir, fosamprenavir, atazanavir, saquinavir, ritonavir, indinavir,nelfinavir, and amprenavir.

In other embodiments, the at least one viral fusion inhibitor includes,but is not limited to a CD4 antagonist, such as soluble CD4 or anantibody that binds to CD4, such as TNX-355, BMS-806; a CCR5 antagonist,such as SCH-C, SCH-D, UK-427,857, maraviroc, vicriviroc, or an antibodythat binds to CCR5, such as PRO-140; a CXCR4 antagonist, such as,AMD3100 or AMD070; or an antagonist of gp41, such as enfuvirtide.

In other embodiments, the at least one viral integrase inhibitorincludes, but is not limited to, raltegravir.

In other embodiments, the at least one glycosidase inhibitor includes,but is not limited to, SC-48334 or MDL-28574.

Reference will now be made in detail to various exemplary embodiments.It is to be understood that the following detailed description isprovided to give the reader a fuller understanding of certainembodiments, features, and details of aspects of the invention, andshould not be interpreted as a limitation of the scope of the invention.

Example 1 Labeling HIV with Azide-Modified Biomolecules

CEMx174 cells were transfected with HIV_(NL4-3) in a T-150 flask and thevirus production was monitored by reverse transcriptase activity untilpeak virus production occurred (usually 7 to 9 days post transfection).Prior to transfection, the CEMx174 cells were spiked with the followingazide-modified biomolecules: 15-azidopentadecanoic acid (50-100 μM),12-azidododecanoic acid (50-100 μM), tetraacetylatedN-azidoacetylgalactosamine (20-40 μM), and tetraacetylatedN-azidoacetyl-D-mannosamine (20-40 μM).

Infected cells were harvested at 12, 24, 72 hours, and 14 days.Harvested cells were isolated and lysed. Cell lysates were then mixedwith the CLICK-IT® detection reagent, tetramethylrhodamine (TAMRA)alkyne and the CLICK-IT® Protein Reaction Buffer Kit (Invitrogen,Carlsbad, Calif.). Cell lysate samples were run on a one-dimensional gelto monitor changes in the azide labeled proteins over time (FIG. 1).

Labeled virus was also obtained from the transfected cells. Morespecifically, virus-containing supernatants were collected and virus waspurified through 20% sucrose as previously described (Graham, D. R. etal., Proteomics 2008, 8:4919-30). The purified virus was then mixed withthe CLICK-IT® detection reagent, tetramethylrhodamine (TAMRA) alkyne andthe CLICK-IT® Protein Reaction Buffer Kit (Invitrogen, Carlsbad,Calif.). Virus samples were run on a one-dimensional gel to revealazide-labeled viral proteins (FIG. 2). Virus levels were normalized p24content and by one-dimensional gel electrophoresis.

No apparent effects of acute or chronic replication of HIV on hostcellular protein modifications were observed (FIG. 1, compare label tocontrols). The azide-modified biomolecules, however, did label viralproteins and permit their detection at the expected molecular weightsfor HIV viral proteins: 55 KDa (gag-myristoylated); 41 KDa(gp41-palmitoylated) and 120 KDa (gp120-N-glycosylated) at 14 days inchronically infected cells (FIG. 1).

Example 2 Inhibiting Infectivity of HIV

To examine the effect of the azide-modified biomolecules on the innatebiology of the virus, the azide-labeled virus from the transfected cellswas isolated and tested in cell infection studies. Unlabeled HIV, at aconcentration 100 times less than the test samples, was used as acontrol. Viral loads were normalized to p24 abundance and virusincubated for 12 hours on a reporter cell line (TZM/BI). TZM/BI is agenetically engineered HeLa cell line that expresses CD4, CXCR4 and CCR5and contains Tat-inducible luciferase and β-Gal reporter genes. Viralinfectivity was determined by measuring cellular luciferase activitieswith two different luciferase reagents. The results using a single cyclereplication system showed that virus labeled with the azide-modifiedbiomolecules, particularly 12-azidododecanoic acid, and to a lesserextent, 15-azidopentadecanoic acid and tetraacetylatedN-azidoacetylgalactosamine, had a profound impact on the infectivity ofthe virus (FIGS. 3 and 4). The level of inhibition of viral entryobserved was comparable to the level of inhibition observed in cellspre-treated with an anti-retroviral agent, such as a fusion inhibitor ora nucleoside analogue.

Example 3 Toxicity Profile

Little to no toxicity has been observed using these azide-modifiedbiomolecules in various eukaryotic cell lines, suggesting that thesecompounds have minimal toxicity profile and supporting their use in atherapeutic setting.

Example 4 Inhibition of Insect Cell Baculovirus Infectivity

Inhibition of Insect Cell Baculovirus Infectivity with Azido Fatty AcidAnalogs:

The BacMam system uses a modified insect virus (baculovirus) as avehicle to efficiently deliver and express genes in mammalian cells. ANuclear-GFP BacMam 2.0 expression system (Invitrogen, C10602) was usedas a model to determine if PTM analog labeling of the virus would affectmammalian cell infectivity. Viruses were labeled with various PTManalogs and used to infect mammalian cells. Infectivity was determinedby the expression of nuclear-GFP, as viral entry into the cell isrequired for expression of GFP protein.

Labeling and Amplification of Nuclear-GFP BacMam 2.0 Virus:

To label, amplify and enrich BacMam viruses with azide/alkyneposttranslational modification (PTM) analogs, 20 ml of Sf9 insect cellsat a concentration of 1.5E6 cells/ml in Sf-900 II SFM insect cell mediawere infected with Nuclear-GFP BacMam 2.0 at a multiplicity of infection(MOI) of 0.1 viruses/cell. Various PTM analogs including palmitic acidazide (15-azidopentadecanoic acid) (Invitrogen, C10265), myristic acidazide (12-azidododecanoic acid)) (Invitrogen, C10268), fucose alkyne(Invitrogen, C10264), ManNAz (tetraacetylatedN-azidoacetyl-d-mannosamine) (Invitrogen, C33366), and Ga1NAz(tetraacetylated N-azidoacetylgalactosamine) (Invitrogen, C33365), inDMSO or 100% ethanol were added to the insect cells at the same time toa final concentration of 50 uM. The cultures were shaken (120 rpm) inthe dark at 27 C for 4 days. The BacMam baculoviruses were harvested bycentrifugation at 1000×g, 15 for minutes. The resulting supernatantswere filtered through a 0.22 um sterile filter into separate sterile,amber, 50 ml conical vials and stored at 4 C.

Characterization of BacMam Virus Production:

To verify, quantitate, and normalize the amount of enriched virusesobtained from insect cell supernatants, samples of each virus were lysedin SDS sample preparation buffer (SPB), sonicated with a probe tipsonicator, and heated at 90 C to completely dissolve the viral proteins.Viral protein concentrations of the lysates were then determined. Virallysate samples were separated by 1D SDS-PAGE and then analyzed byWestern blot using antibodies against virus specific proteins, gp64[eBiosciences, mouse monoclonal, 14-69995-85) and VSV-G (Sigma, rabbitpolyclonal, V4888)]. Relative virus concentrations obtained from proteinanalysis and Western blotting were used to normalize virus addition insubsequent viral transduction experiments.

Mammalian Cell Infection Protocol:

To determine if the PTM analog-labeled viruses retain the ability toinfect mammalian cells, 50,000 U2-OS cells (human osteosarcoma cellline) were plated onto a 6 well-chamber glass bottom plate. 20 or 50 uLof enriched virus was added into 2 ml final volume of media+serum(McCoy's+10% FBS). The plates were then incubated overnight at 37 C, in5% CO₂. The following day cells were imaged on AMG EVOS fluorescencemicroscope at 10× or 20× magnification using both white light and GFPfilters (FIG. 5).

To determine the effect of PTM analog incorporation on the ability ofBacMam to enter mammalian cells, a BacMam construct that expressesnuclear GFP was used. As BacMam virus reproduces in insect cells, butnot in mammalian cells, sugar and fatty acid analog-labeled viruses wereproduced in SF9 insect cells, and the labeled viruses were then used todetermine infectivity in U2-OS cells (human osteosarcoma cell line).Nuclear GFP expression in U2-OS cells can only take place if the virusis able to enter the cell. The panels of FIG. 5 show both phase (bottomrow of panels) and fluorescent GFP images (top row of panels) of U2-OScells infected with PTM analog-labeled BacMam viruses. In these panels,cells treated with myristate-azide and palmitate-azide labeled virusesshowed no nuclear-GFP expression, while control cells (no virus) andcells treated with sugar-labeled viruses show significant nuclear GFPexpression.

What is claimed:
 1. A method of producing a virus labeled with anazide-modified fatty acid, an azide-modified carbohydrate, anazide-modified isoprenoid lipid, or a pharmaceutically acceptable saltthereof, the method comprising contacting a cell infected with the viruswith the azide-modified fatty acid, the azide-modified carbohydrate, theazide-modified isoprenoid lipid, or pharmaceutically acceptable saltthereof so that the azide-modified fatty acid, the azide-modifiedcarbohydrate, the azide-modified isoprenoid lipid, or pharmaceuticallyacceptable salt thereof enters the cell and is incorporated into aprotein of the virus, thereby producing the labeled virus.
 2. The methodof claim 1, wherein the azide-modified fatty acid or pharmaceuticallyacceptable salt thereof, has the formula:Y—CH₂—X—CO₂H wherein, Y is H or an azido group; and when Y is an azidogroup, X is a linear or branched carbon chain comprising 6 to 28carbons, wherein one or more of said carbons may be independentlyreplaced by an oxygen, selenium, silicon, sulfur, SO, SO₂ or NR₁, orwherein one or more pairs of said carbons adjacent to one another may beattached to one another by a double or triple bond; or when Y is H, X isa linear or branched carbon chain comprising 6 to 28 carbons, whereinone hydrogen on one of said carbons is replaced with an azido group andwherein one or more of said carbons not having an the azido groupattached thereto may be independently replaced by an oxygen, selenium,silicon, sulfur, SO, SO₂ or NR₁, or wherein one or more pairs of saidcarbons adjacent to one another and not having an azido group may beattached to one another by a double or triple bond; wherein, R₁ is H oran alkyl comprising 1 to 6 carbons.
 3. The method of claim 2, wherein Yis an azido group.
 4. The method of claim 3, wherein X is a linearcarbon chain.
 5. The method of claim 4, wherein the carbon chaincomprises 8 to 15 carbons.
 6. The method of claim 5, wherein the carbonchain does not contain an oxygen, selenium, silicon, sulfur, SO, SO₂ orNR₁.
 7. The method of claim 6, wherein the carbon chain does not containa double or triple bond.
 8. The method of claim 7, wherein theazide-modified fatty acid is 15-azidopentadecanoic acid orpharmaceutically acceptable salt thereof.
 9. The method of claim 7,wherein the azide-modified fatty acid is 12-azidododecanoic acid orpharmaceutically acceptable salt thereof.
 10. The method of claim 1,wherein the cell is a human cell.
 11. The method of claim 1, wherein thevirus is a human immunodeficiency virus.
 12. The method of claim 1,wherein the cell is an insect cell.
 13. The method of claim 1, whereinthe virus is a baculovirus.
 14. The method of claim 1, wherein theazide-modified carbohydrate, azide-modified fatty acid, azide-modifiedisoprenoid lipid, or pharmaceutically acceptable salt is formulated witha pharmaceutically acceptable excipient.
 15. The method of claim 14,further comprising the step of administering to the cell theazide-modified carbohydrate, azide-modified fatty acid, azide-modifiedisoprenoid lipid, or pharmaceutically acceptable salt which isformulated with a pharmaceutically acceptable excipient.
 16. A method oftracking a virus in vivo comprising the steps of contacting culturedcells or a subject with an azide-modified carbohydrate, azide-modifiedfatty acid, azide-modified isoprenoid lipid, or a pharmaceuticallyacceptable salt thereof; contacting the cultured cells or the subjectwith an alkyne labeled reporter molecule; and tracking thereporter-labeled virus in the cultured cells or the subject.
 17. Themethod of claim 16, wherein the cultured cells or the subject iscontacted with an azide-modified fatty acid or pharmaceuticallyacceptable salt thereof.
 18. The method of claim 17, wherein theazide-modified fatty acid or pharmaceutically acceptable salt thereof,has the formula:Y—CH₂—X—CO₂H wherein, Y is H or an azido group; and when Y is an azidogroup, X is a linear or branched carbon chain comprising 6 to 28carbons, wherein one or more of said carbons may be independentlyreplaced by an oxygen, selenium, silicon, sulfur, SO, SO₂ or NR₁, orwherein one or more pairs of said carbons adjacent to one another may beattached to one another by a double or triple bond; or when Y is H, X isa linear or branched carbon chain comprising 6 to 28 carbons, whereinone hydrogen on one of said carbons is replaced with an azido group andwherein one or more of said carbons not having an the azido groupattached thereto may be independently replaced by an oxygen, selenium,silicon, sulfur, SO, SO₂ or NR₁, or wherein one or more pairs of saidcarbons adjacent to one another and not having an azido group may beattached to one another by a double or triple bond; wherein, R₁ is H oran alkyl comprising 1 to 6 carbons.
 19. The method of claim 18, whereinY is an azido group.
 20. The method of claim 19, wherein X is a linearcarbon chain.
 21. The method of claim 20, wherein the carbon chaincomprises 8 to 15 carbons.
 22. The method of claim 21, wherein thecarbon chain does not contain an oxygen, selenium, silicon, sulfur, SO,SO₂ or NR₁.
 23. The method of claim 22, wherein the carbon chain doesnot contain a double or triple bond.
 24. The method of claim 23, whereinthe azide-modified fatty acid is 15-azidopentadecanoic acid orpharmaceutically acceptable salt thereof.
 25. The method of claim 23,wherein the azide-modified fatty acid is 12-azidododecanoic acid orpharmaceutically acceptable salt thereof.